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List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Tranell, A., Fenyö, E M., Schwartz, S. (2010) Serine- and Argininerich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of
HIV-1 vpr mRNA by Inhibiting the 5´-Splice site of Exon 3. Journal
of Biological Chemistry. 285:31537-31547.
II
Tranell, A., Fenyö, E M., Tingsborg, S., Schwartz, S. (2011) Inhibition of Splicing by Serine-Arginine Rich Protein 55 (SRp55) Causes
the Appearance of Partially Spliced HIV-1 mRNAs in the Cytoplasm. V irus Research. 157:82-91.
III Tranell, A., Fenyö, E M., Schwartz, S. Indole-related Compounds
from the National Cancer Institute (USA) Repository do not Inhibit
HIV-1 Replication in Primary Blood Mononuclear Cells. Manuscript.
Reprints were made with permission from the respective publishers.
Contents
Introduction ................................................................................................... 11 Viruses ...................................................................................................... 11 Retroviruses .............................................................................................. 12 Human immunodeficiency virus .............................................................. 12 AIDS .................................................................................................... 13 Treatment of HIV-1 infection .............................................................. 14 HIV-1 genomic organization ............................................................... 15 Virion structure and infectious cycle ................................................... 18 Post-transcriptional processing ................................................................. 21 Pre-mRNA splicing .............................................................................. 22 SR proteins ........................................................................................... 27 HIV-1 pre-mRNA splicing .................................................................. 30 Antiviral therapy and HIV-1 splicing .................................................. 34 The present investigation .............................................................................. 35 Aims of the study ...................................................................................... 35 Experimental system................................................................................. 35 Results ...................................................................................................... 36 Paper I .................................................................................................. 36 Paper II ................................................................................................. 38 Paper III ............................................................................................... 39 Concluding remarks and future prospects ..................................................... 41 Acknowledgements ....................................................................................... 44 References ..................................................................................................... 47 Abbreviations
AIDS
ALV
AZT
CA
CTD
DNA
ELISA
ER
ESE
ESS
FI
GAR
HAART
HIV
HIV-1
HIV-2
hnRNP
IN
ISE
ISS
LTR
MA
mRNA
NAb
NC
NNRTI
NRTI
PBMC
PCR
PPT
PR
Py
RNA
RRM
RS
RT
acquired immunodeficiency syndrome
avian leucosis virus
azidothymidine
capsid
carboxy-terminal domain
deoxyribonucleic acid
enzyme-linked immunosorbent assay
endoplasmic reticulum
exonic splicing enhancer
exonic splicing silencer
fusion inhibitor
guanosine-adenosine-rich
highly active antiretroviral therapy
human immunodeficiency virus
human immunodeficiency virus type 1
human immunodeficiency virus type 2
heterogeneous nuclear ribonucleoprotein
integrase
intronic splicing enhancer
intronic splicing silencer
long terminal repeat
matrix
messenger ribonucleic acid
neutralizing antibody
nucleocapsid
non-nucleoside reverse transcriptase inhibitor
nucleoside reverse transcriptase inhibitor
primary blood mononuclear cell
polymerase chain reaction
polypyrimidine tract
protease
polypyrimidines
ribonucleic acid
RNA recognition motif
arginine-serine-rich domain
reverse transcriptase
SA
SD
SIV
snRNA
snRNP
SR protein
SRPK
TAR
U2AF
UNAIDS
U snRNA
U snRNP
splice acceptor (3´ splice site)
splice donor (5´ splice site)
simian immunodeficiency virus
small nuclear ribonucleic acid
small nuclear ribonucleoprotein
serine-arginine rich protein
SR-specific protein kinase
transactivation response region
U2 snRNP auxiliary factor
the Joint Nations Programme on HIV/AIDS
uridine-rich small nuclear RNA
uridine-rich small nuclear RNP
Introduction
According to the Joint Nations Programme on HIV/AIDS (UNAIDS) there
were 34 million people living with human immunodeficiency virus (HIV)
infection at the end of 2010. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS) and the number of people in 2010 dying of
AIDS-related causes was 1.8 million. Thanks to changes in behavior and
antiretroviral therapy the epidemic is declining in most countries. Apart from
faster and broader treatments together with the spread of information there is
also a constant need for new approaches and antivirals to attack the virus. In
order to develop new strategies, we need to know more about the virus itself,
its life cycle and the way it behaves in our cells. We also need to have more
knowledge about how the cells and our immune system react during an HIV
infection. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is the main
cause of AIDS and therefore this thesis is focusing on HIV-1. The splicing
process of HIV-1 pre-mRNA is highly regulated and a prerequisite for the
virus to be infectious. In this thesis I have therefore studied the regulation of
HIV-1 pre-mRNA splicing, with the hope that these findings should open up
new possibilities in the fight against the virus.
Viruses
A virus is a contagious material that can only replicate inside a cell. It is
debated whether viruses should be considered as living or non-living. The
first virus to be discovered was the tobacco mosaic virus. It was found to be
the causative agent of tobacco mosaic disease in 1892 by Dimitri Ivanovsky
and independently by Martinus Beijerinck in 1898. Bacteria had already
been discovered by that time. And the tobacco mosaic virus was stated by
Beijerinck to be an agent that passed filters which normally trapped bacteria
and that the agent could multiply within living plants. At the same time footand-mouth disease was found to be caused by an agent that could not be
retained in filters, an observation made by Friedrich Loeffler and Paul
Frosch. Together with the observation that both the foot-and-mouth disease
and the tobacco mosaic disease causing agents were unable to grow in solutions where known bacteria could multiply, this made the researchers come
to the conclusion that the agents must be something other than bacteria.
(Flint and American Society for Microbiology., 2009).
11
In the beginning researchers thought that this new agent was a liquid. But
they were described as particles after d´Herelle´s plaque assay in 1917 and
the first electron micrographs of tobacco mosaic virus in 1939. The first
human virus to be discovered was the yellow fever virus, by Walter Reed in
1901 (Lustig and Levine, 1992).
Ever since these discoveries 40 000 viruses have been classified, with varieties in size, shape, genome, host cell and life cycle. In addition, studies of
viruses have given us knowledge not only about the viruses themselves, but
also about the cellular machinery and processes like pre-mRNA splicing and
gene silencing.
Retroviruses
One class of viruses that can reverse transcribe their RNA genome into DNA
is called retroviruses, and belongs to the family Retroviridae. A retrovirus
has the enzyme reverse transcriptase incorporated into the virion. Another
enzyme incorporated into the virion is integrase that mediates integration of
viral DNA into the host genome (Flint and American Society for
Microbiology., 2009). Retroviral genomes are usually 6–11 kb in size and
vary in complexity. The simplest retroviruses only have genes encoding the
virion proteins, i.e. gag, pol and env, while the more complex retroviruses
additionally have genes encoding accessory proteins that affect viral gene
expression, fine tune replication and make it possible for the virus to adapt to
host. These proteins could also be required in vivo. All retroviral genomes
have LTRs (long terminal repeats) in their 5´- and 3´-end, where the promoter and sequences for polyadenylation are positioned (Vogt, 1997b).
Retroviruses were discovered at the beginning of the last century. These
retroviruses were associated with disease in chickens and were subsequently
named avian leucosis virus (ALV) and Rous sarcoma virus. Ever since then
more and more retroviruses have been discovered, some of which infect
humans (Vogt, 1997a). One of these viruses, the human immunodeficiency
virus (HIV), was discovered in the beginning of 1980s and was later found
to cause AIDS.
Human immunodeficiency virus
HIV is an enveloped retrovirus belonging to the retroviral sub-family lentivirus. There are two types of HIV, type 1 and type 2. HIV-2 is rather rare and
originates from simian immunodeficiency virus (SIV) that infects the sooty
mangabey (Cercocebus atys). HIV-1 is more common than HIV-2 and originates from SIV that infects chimpanzees, called SIVcpz. There are three
different sub-groups of HIV-1: M, N and O, which represent transmissions
12
of SIVcpz to humans on three separate occasions. Sub-group M is the most
widespread and is the cause of more than 95% of all HIV-1 infections. This
group is further divided into subtypes A-K. Phylogenetic analyses date the
common ancestor of the subtypes in the group M between 1902 and 1921.
(Gao et al., 1999).
HIV is spread via sexual contact, blood and from mother to child during
birth or via breast milk. The first transmission of SIV to humans occurred,
probably by exposure to chimpanzee blood, in central Africa. The SIV
strains in infected chimpanzees are closest related to HIV-1 group M that has
been found in southeastern Cameroon. One theory is that HIV-1 infection
spread from there to the great city of Leopoldville (Kinshasa) in Belgian
Congo (Democratic Republic of the Congo) where the pandemic could start
(Worobey et al., 2008). The spread of the virus was initially rather slow.
However during the 1950s and 1960s the epidemic increased. This was
probably due to great changes in Africa, with growing cities and the introduction of vaccine programs (reuse of needles). There was also increased
travel to and from Africa and a sexual revolution may also have contributed
to the spread of HIV (Sharp and Hahn, 2008).
At the end of 2010 34 million people were living with an HIV infection,
but the total number is falling compared to the peak in 1997. Of all people
infected, 68% are living in Sub-Saharan Africa, a region with only 12% of
the global population. The epidemic is most severe in southern Africa. Even
though the total number of infected people is decreasing, there is an increase
in some parts of the world. For instance in Eastern Europe and Central Asia,
there was a 250% increase in the number of infected people from 2001 to
2010 (UNAIDS, 2011).
AIDS
HIV is the causative agent of acquired immunodeficiency syndrome (AIDS)
- the state where the immune system is degraded and the host easily acquires
opportunistic infections. The first case of AIDS was described in 1981 and
about two years later the causative agent of AIDS was found to be HIV. The
main discoveries took place in two laboratories: Gallo in Bethesda, Maryland USA and Montagnier at the Pasteur Institute, Paris France (Gallo and
Montagnier, 2003, Wainberg and Jeang, 2008). AIDS, caused by HIV, kills
more people than any other infectious disease (Flint and American Society
for Microbiology., 2009).
In an untreated patient AIDS usually occurs within 8-10 years after the initial HIV infection. AIDS is the state where the numbers of CD4+ T cell
counts are below 200 per ml blood (normal count is around 1000 per ml
blood) and the numbers of CD8+ T cell counts also decreases. The decrease
in immune cells leads to conditions such as cancer (e.g. Kaposi’s sarcoma
and B cell lymphomas), oral lesions, basal cell carcinoma and bacterial, fun13
gal and viral infections as well as neurological disorders such as AIDS dementia and meningitis. AIDS-related deaths in 2010 were estimated to 1.8
million. Due to increased treatment and changed behavior this number has
been decreasing over the last years throughout the world apart from Eastern
Europe and Central Asia (UNAIDS, 2011).
Treatment of HIV-1 infection
Antiviral therapy
The first treatment for HIV infection was azidothymidine (AZT), approved
for marketing in 1987 (Mitsuya et al., 1985). There have been several antiviral drugs tested on humans and today more than 20 drugs have been approved for HIV infection treatment. The main targets for HIV drugs are Reverse transcriptase (RT), Protease (PR), Integrase (IN) and virus-cell interaction (attachment, fusion and entry). The RT inhibitors are either nucleoside
(NRTI) or non-nucleoside reverse transcriptase inhibitors (NNRTI). The PR
inhibitors (PIs) mimic the peptide linkage in the polyprotein gag-pol. The
drugs targeting IN inhibit the integration process of the viral genome into the
host genome (De Clercq, 2007). Virus-cell interaction inhibitors like fusion
inhibitors (FIs) prevent the virus from entering the cell (Pugach et al., 2008).
One approach to treating HIV infections is the HAART (highly active antiretroviral therapy) where the patients are treated with a combination of at
least three anti-HIV drugs, leading to a lower risk of drug resistance.
The HIV genome is highly mutable due to the lack of proofreading activity of the reverse transcriptase. Mutations may lead to drug resistant viruses
and there is a constant need of new types of therapy. Another problem in
clearance of the infection is the integration of viral DNA into the host genome and no treatment available today can clear an infection. A third problem is the asymptomatic phase (with viral replication) during the first period
of infection. Taken together, all these factors create a constant need for new
antivirals and new strategies to discover infections, which demands further
investigation of the virus life cycle.
Vaccine
Ever since HIV was discovered researchers have been trying to develop a
vaccine in order to stop the epidemic. The major issues in designing a vaccine against HIV-1 are the genetic heterogeneity of the virus (due to the high
mutation rate, RNA recombination and immune selection), the integration of
the viral DNA into the host genome, virus can spread through cell-cell interaction, virus infects areas of reduced immune surveillance, the infection
compromises immune function. Another issue is the ability of the virus to
evade the immune system by ‘resting’ in lymphocytes and macrophages.
Another problem is that a vaccine would have to protect against millions of
14
variants of virus particles and so far there is no vaccine available that can
protect against HIV infection (Douek et al., 2006). A number of clinical
trials have been performed with different vaccine candidates. One study was
performed where researchers evaluated priming injections of recombinant
canarypox HIV-vector vaccine (ALVAC-HIV) and booster injections of a
recombinant gp120 envelope protein (AIDSVAX B/E) (Rerks-Ngarm et al.,
2009). The vaccine showed a 31.2% efficacy but it did not have any effect
on early viral load or the CD4 T cell count in HIV-1 infected patients. However, the study may provide new insight into the development of vaccines
(Nossal, 2011). Another field in vaccine research is the production of neutralizing antibodies (NAbs). These studies mainly focus on antibodies
against the viral envelope protein gp120. One prototype of a NAb called
VRC01 has been shown to neutralize 90% of highly diverse HIV isolates
(Walker and Burton, 2010, Wu et al., 2010, Zhou et al., 2010). A development of an efficient vaccine will need more knowledge about the viral life
cycle as well as the immune response during an HIV-1 infection.
HIV-1 genomic organization
The genome of HIV-1 is approximately 9 kilobases (kb) in size and contains
the genes: gag-pol, tat, rev, nef, vpr, vpu, vif and env (Fig. 1). As will be
described later, the HIV-1 genome can produce mRNAs that are differently
spliced. These mRNAs can be classified into unspliced (9 kb), partially
spliced (4 kb) and fully spliced (2 kb). In the 5´- and 3´-ends are the Long
Terminal Repeats (LTRs) that contain sequences for transcription and polyadenylation.
15
tat
gag
rev
env
vif
vpr
pol
nef
vpu
LTR
LTR
1
2
3
4
7
2a
3a
4c
4a
4b
5
2aE
3aE
4E
4cE
4aE
4bE
5E
Figure 1.Schematic representation of the HIV-1 genome. Boxes represent open reading frames, circles indicate splice donors and triangles indicate splice acceptors.
Black bars represent the different exons (Schwartz et al., 1990a).
Gag-Pol
The gag-pol gene encodes two polyprotein precursors, Gag and Gag-Pol.
The Gag polyprotein gives rise to the matrix (MA), capsid protein (CA),
nucleocapsid protein (NC) and p6, and the Gag-Pol polyprotein gives rise to
protease (PR), reverse transcriptase (RT) and integrase (IN). The gag-pol
mRNA is the same molecule as the pre-mRNA and may also be incorporated
into the virion and serve as genome (Sierra et al., 2005).
Tat
The tat gene encodes the Tat protein that is essential for viral replication.
Transcription from the viral promoter LTR is rather inefficient but in the
presence of Tat, transcription is enhanced about 100-fold. Tat binds to an
RNA hairpin in the 5´-end of the transcript called the transactivation re16
sponse region (TAR). The increase of transcription is probably due to enhanced phosphorylation of RNA polymerase II C-terminal domain (CTD),
which increases the elongation of RNA transcription (Frankel and Young,
1998). The increase in elongation is due to Tat association with the elongation factor P-TEFb, and recruitment of P-TEFb to the viral promoter increases the elongation (Zhu et al., 1997). Tat may also be involved in regulation
of viral splicing, see the section HIV-1 pre-mRNA splicing.
Rev
The Rev protein is translated from a fully spliced (2 kb) mRNA. After translation, Rev enters the nucleus where it binds to the Rev Response Element
(RRE) on the partially spliced (4 kb) and unspliced (9 kb) HIV-1 mRNAs
and thereby facilitates their export to the cytoplasm by interacting with Crm1
and other cellular factors (Nekhai and Jeang, 2006, Pollard and Malim,
1998).
Nef
The name of the Nef protein comes from negative factor because it was originally thought of as negatively affecting viral transcription but Nef has been
found to enhance the infectivity of HIV-1. The enhancement is caused by
down regulating the number of CD4 receptors on the cell surface to facilitate
the release of new virions. Nef can also down regulate Major Histocompatibility Complex type I (MHC I), to prevent detection by the cytotoxic T cells
(Benson et al., 1993, Schwartz et al., 1996).
Env
The env gene encodes the proteins gp120 (SU) and gp41 (TM) that sits in
the envelope membrane. They are produced through cleavage of the Env
polyprotein gp160 by cellular proteases. SU and TM are involved in entry
and fusion between viral and cellular membranes. gp120 interacts with the
CD4 receptor on T cells and macrophages and thereafter undergoes a conformational change that facilitates the interaction between gp120 and its coreceptor (CXCR4 and CCR5). Thereafter, gp41 acts as a fusion protein, mediating viral entry (Freed, 2001).
Vpu
The integral membrane viral protein U (Vpu) induces degradation of the
CD4 receptor in the endoplasmic reticulum (ER). Vpu can also facilitate
virion release (Bolduan et al., 2011, Bour and Strebel, 2003, Margottin et al.,
1998, Schubert et al., 1998, Willey et al., 1992).
17
Vif
The viral infectivity factor (Vif) is required for infectivity of HIV-1 by inducing degradation of the innate antiviral response APOBEC3G and inhibition of its virion incorporation. Without Vif, the APOBEC3 enzymes deaminate C to U residues in the HIV-1 minus strand DNA produced by RT, causing G to A mutations in the proviral DNA reducing the infectivity of the
virus (Gabuzda et al., 1992, Lecossier et al., 2003, Marin et al., 2003,
Sheehy et al., 2002). It has also been reported that Vif is present in the virion
and that it facilitates the reverse transcription process (Cancio et al., 2004,
Carr et al., 2008).
Vpr
The viral protein R (Vpr) is a 96 amino acid, 14 kDa protein. It was isolated
in 1990 and is highly conserved (Cohen et al., 1990, Planelles et al., 1996,
Yuan et al., 1990). Vpr facilitates infection by mediating the nuclear import
of HIV-1 DNA in non-dividing cells. This is achieved by Vpr functioning as
a component of the pre-integration complex that interacts with the nuclear
pore complex (Le Rouzic et al., 2002, Nie et al., 1998). Vpr also induces cell
cycle arrest in the G2 phase by promoting phosphorylation of mitosis checkpoint proteins. This may result in apoptosis of infected cells and transactivation of the viral promoter LTR (Dehart and Planelles, 2008, He et al.,
1995, Roshal et al., 2001). Arrest in G2 could also be caused by Vpr-induced
breaks in the nuclear lamina structure, causing the cell nuclei to rapture (de
Noronha et al., 2001). Vpr can also function as a transactivator of LTR by
interacting with transcription factor IIB (TFIIB) and by facilitating the activation of LTR by p300 (Agostini et al., 1996, Felzien et al., 1998). In addition, there are several reports that suggest that Vpr aids in evasion of the
immune response. This appears to occur by induction of cell death in immune system cells (Kogan and Rappaport, 2011).
Virion structure and infectious cycle
HIV-1 is an enveloped virus that carries two copies of the genomic RNA (+)
strand within its virion. The RNA molecules are coated with nucleocapsid
protein (NC). The virion also contains 50 to 100 molecules of reverse transcriptase (RT), 50 to 100 copies of integrase (IN), protease (PR) as well as
cellular RNAs, e.g. tRNA. Viral proteins Vif and Vpr are also present in the
virion. The capsid protein (CA) together with the matrix protein (MA) constitutes the viral building blocks (Flint and American Society for
Microbiology., 2009) (Fig. 2).
18
Vpr
Vif
Reverse
transcriptase
Envelope
Protease
Capsid
Integrase
Matrix
Nucleocapsid proteins
and genomic RNA
Figure 2. The HIV-1 virion containing capsid, matrix, envelope, integrase, protease,
reverse transcriptase, Vpr, Vif, nucleocapsid proteins and genomic RNA.
Attachment and entry
HIV-1 enters the cell after binding with the viral gp120 envelope protein to
the cellular CD4 receptor present on T cells, monocytes, macrophages and
dendritic cells (Fig. 3). The viral protein gp120 thereafter interacts with the
co-receptor CXCR4 or CCR5. This leads to fusion, mediated by gp41, between the viral envelope and the plasma membrane. The viral core, consisting of viral RNA genome (coated with NC protein), integrase (IN), reverse
transcriptase (RT), matrix (MA) and Vpr is released into the cytoplasm of
the cell (Frankel and Young, 1998, Hernandez et al., 1996).
Reverse transcription and integration
In the pre-integration complex, RT reverse transcribes the viral RNA moleLys
cules. This process is initiated by a cellular tRNA primer and results in (–)
DNA in a 3´ to 5´ direction. Following transcription, the RNase H of RT
degrades the template RNA. The (+) DNA strand is transcribed using the
first DNA strand as a template and viral RNA as a primer. The DNA together with IN and other proteins are called the pre-integration complex. This
complex is localized to the nucleus by Vpr and imported via the nuclear pore
complex, a mechanism that is still rather unclear. In the nucleus, the viral
protein IN catalyzes the insertion of viral DNA into the host cell genome.
Nicking of the two viral DNA-ends starts the integration. The new 3´-ends
are then joined to the target DNA. Cellular enzymes repair the resulting gaps
19
within the DNA. When integrated into the host genome the viral DNA is
referred to as the provirus and behaves as a cellular gene (Freed, 2001,
Turner and Summers, 1999).
Transcription, pre-mRNA processing and translation
Transcription of the HIV-1 genome is carried out by RNA polymerase II and
is enhanced by the viral protein Tat. The promoter is positioned in the LTR
region that contains the three regions U3, R and U5. The promoter and enhancer sequences are located in U3 and transcription starts from the R region. The cellular machinery initially transcribes at a low level. Transcription of the HIV-1 genome is dependent on cellular factors, especially NF-κb,
which in T cells is only active in activated cells. Transcription may be enhanced as much as 100-fold by the viral protein Tat that recognizes a secondary structure in the 5´-end of the RNA, called transactivation response
(TAR). Tat interacts with cellular factors, which leads to phosphorylation of
the C-terminal domain of RNA Polymerase II, which stimulates the transcription by enhancing the elongation (Frankel and Young, 1998, Zhu et al.,
1997).
The primary, full-length transcript encodes Gag and Gag-Pol proteins and
also serves as the viral genome in newly synthesized virions. The other HIV1 proteins are produced from mRNAs that have been alternatively spliced
from the full-length transcript. The only mRNAs that are immediately exported to the cytoplasm are the fully spliced 2 kb mRNAs. The Tat and the
Nef proteins are products of the 2 kb mRNAs. Another protein produced
from 2 kb mRNAs is Rev, which facilitates the export of the full-length transcript and the 4 kb mRNAs. Full-length viral mRNAs are translated into Gag
and Gag-Pol polyproteins that localize to and assemble at the cell membrane
together with the viral genomic RNA, Vpr, Vif and Nef. The 4 kb mRNAs
encode for the Env polyprotein that is synthesized and modified in the endoplasmic reticulum (ER) and Golgi apparatus to produce gp120 and gp41
(Arya and Gallo, 1986, Feinberg et al., 1986, Nekhai and Jeang, 2006,
Pollard and Malim, 1998, Schwartz et al., 1990a, Schwartz et al., 1990b).
Assembly, budding and maturation
The viral protein Vpu assists the transport of the envelope proteins to the cell
membrane by promoting degradation of newly synthesized CD4 in the ER
(Dube et al., 2010). The cell surface becomes coated with the envelope proteins, which are prevented from binding to cell surface CD4 receptors
through degradation of CD4 mediated by the viral protein Nef (Frankel and
Young, 1998). Gag molecules associate with the plasma membrane and the
RNA genome and assembly initiates. The virus assembles and buds from the
infected cell as a noninfectious, immature virus particle. After budding, the
Gag-Pol polyprotein is self-cleaved into RT, IN and PR. The Gag polypro-
20
tein is cleaved by the protease into matrix, capsid, nucleoprotein and p6 and
the virion becomes mature (Gottlinger, 2001).
Virus particle
1
10
CD4 receptor
2
T-cell/Macrophage
9
3
4
5
6
7
8
Cell nucleus
Figure 3. Schematic representation of the HIV-1 infectious cycle. 1) Virus attachment to CD4 receptor, fusion is mediated by binding to co-receptor. 2) Virus entry
and core release. 3) Following reverse transcription proviral DNA is imported to the
nucleus and 4) integrated into the host genome. 5) Viral DNA is transcribed and
spliced by cellular machinery. 6) Fully spliced mRNAs are transported to the cytoplasm and translated. 7) The viral protein Rev returns to the nucleus and 8) mediates
the export of partially spliced and unspliced mRNAs, which can be translated in the
cytoplasm. 9) Unspliced mRNA is transported to the cell membrane, where Env,
Gag and Gag-Pol polyproteins have assembled, and incorporates into new virus
particles as the viral genome. 10) The new virus particle is released and after proteolytic cleaving it becomes mature.
Post-transcriptional processing
DNA is transcribed into mRNA, which will later be translated into protein.
Before translation can occur several different modifications of the premRNA have to take place. These modifications occur co-transcriptionally
and include i) the addition of a 7-methyl guanosine (m7G) cap at the 5´-end
of the RNA ii) the production of a poly(A) tail by cleavage and polyadenylation of the 3´-end of the pre-mRNA iii) as well as excision of introns by the
process called splicing (Beyer and Osheim, 1988, Maniatis and Reed, 2002,
Tennyson et al., 1995). The 5´-cap and the 3´-poly(A) tail have diverse functions: They protect the mRNA from degradation and facilitate translation by
easing the export of mRNA, and in the cytoplasm they promote translation
21
(Ford et al., 1997, Furuichi et al., 1977, Wakiyama et al., 2000). This thesis
focuses on HIV-1 pre-mRNA splicing. The following section therefore gives
a detailed description of this process.
Pre-mRNA splicing
Splicing is the process where the exons in the pre-mRNA are joined together
and the introns in between are excised. In 1993 Philip Sharp and Richard
Roberts were awarded the Nobel Prize for their discovery of splicing.
The biochemical process of splicing can be divided into two transesterification steps (Fig. 4). The first step is when the phosphodiester bond between
the first exon and the 5´-end of the downstream intron is broken resulting in
a free exon and a lariat RNA intermediate, consisting of the intron and the
second exon. In the second step the phosphodiester bond between the second
exon and the 3´-end of the intron is broken, the intron is excised and the two
exons are joined together. A prerequisite for the splicing reaction to occur is
the assembly of the spliceosome, which is partly directed by the recognition
of sequences in the 5´ splice site also called splice donor (5´-end of intron),
3´ splice site also called splice acceptor (3´-end of intron), a polypyrimidine
tract and a branch point (Burge, 1999). The 5´ splice site consensus sequence
is AG/GURAGU (intron part is underscored) and is a binding site for U1
snRNP. The 3´ splice site contains the conserved sequence YAG/N and a
polypyrimidine tract (PPT) (Py) upstream. The 3´ splice site is the binding
site for the cellular splicing factor U2AF, where the subunit U2AF65 binds
to the PPT and the subunit U2AF35 binds to the AG sequence. Upstream of
the PPT is the branch point, YNYURAC that is bound by the branch point
binding protein SF1/mBBP (Graveley, 2000).
22
5’ splice site
3’ splice site
branch point
Pre-mRNA
Exon 1 GU
PPT
A
Ex
on
A
Exon 1 Exon 2
mRNA
1
(Py)
AG Exon 2
OH
AG Exon 2
A
Intron lariat
Figure 4. Schematic representation of the two transesterification steps occurring
during pre-mRNA splicing. Boxes represent exons and lines represent introns. The
branch point (BP), polypyrimidine tract (Py), 5´ (GU) and 3´ (AG) splice sites are
indicated.
The initial step of splicing in mammalian cells is the exon definition, a process in which factors at the 5´ splice site and the 3´ splice site interact. This
interaction causes the assembly of the spliceosome into the early splicing
complex, called E complex (Hoffman and Grabowski, 1992, Robberson et
al., 1990). Splicing is not only regulated by transacting factors, but also by
cis-elements within the pre-mRNA. These elements are called Exonic splicing enhancers, ESEs, Exonic splicing silencers, ESSs, Intronic splicing enhancers, ISEs, and Intronic splicing silencers, ISSs, which all recruit activators or repressors of splicing (Maniatis, 1991). Generally, many enhancer
sequences recruit members of the serine-arginine rich (SR) protein family,
which will be described further on, and most silencer sequences recruit
members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family
(Dreyfuss et al., 1993, Graveley, 2000, Pozzoli and Sironi, 2005), but this is
by no means universally true. Splicing can be either constitutive, i.e. exons
are always spliced or excessed in the final mRNA, or alternative, i.e. exons
23
are joined together in multiple ways. Because HIV-1 uses alternative splicing the main focus in this thesis will be on alternative splicing, which will be
explained further on.
The spliceosome
The spliceosome (i.e. U2-dependent spliceosome) (Fig. 5) contains four
small nuclear ribonucleoprotein particles (snRNPs U1, U2, U4/U6 and U5)
and several non-snRNPs like serine-arginine rich (SR) proteins. The U1
snRNP binds to the 5´ splice site, thereafter U2 snRNP auxiliary factor
(U2AF) binds to the PPT and to the 3´-terminal AG, and splicing factor 1
(SF1) binds to the branch point. This leads to the formation of the E complex. These interactions promote the association of U2 snRNP with the
branch point to form the ATP-dependent A complex. After U2 snRNP binding, U4/U6 snRNP and U5 snRNP association form the B complex. Further
progress in the splicing reaction requires destabilization of the U4 snRNA
carried out by RNA helicases. This step will create the catalytically active
spliceosome, the C complex, which catalyzes the two chemical steps of
splicing (Burge, 1999, Chen and Manley, 2009). There is also a minor class
of spliceosome called the U12-dependent spliceosome but the work in this
thesis is based on the U2-dependent spliceosome and the U12 will therefore
not be presented any further.
24
5’ splice site
3’ splice site
branch point
Pre-mRNA
YNYURAC
AG GURAGU
U1
E complex
(Py) YAG N
U2AF
GU
A
AG
U2 U2AF
A
AG
U1
A complex
PPT
GU
U6
U4
U5
U1
B complex
U4
U6
U2 U2AF
U5
GU
A
AG
U4
U1
U5
U6
C complex
U2
U2AF
A
mRNA
Intron lariat
Figure 5. Schematic representation of the splicing process. Splicing regulatory sequences (5´ splice site, 3´ splice site, branch-point and polypyrimidine tract) within
the pre-mRNA are indicated in the upper panel. Boxes represent exons and thin line
represents intron. The spliceosome assembly is indicated stepwise in the lower panel, from the formation of E complex to the formation of the catalytic C complex.
Factors involved in the assembly are indicated: U2AF and U snRNPs (U1, U2, U4,
U5 and U6). The final products in the process are mRNA and intron lariat, shown in
the bottom of the panel.
25
Alternative splicing and splice site recognition
Most pre-mRNAs in higher eukaryotes contain multiple introns making it
possible to create different mature mRNAs from the same pre-mRNA by
alternative splicing patterns. Alternative splicing was first described in 1980
and since then we have come to understand the diversity of the expression
from eukaryotic genes (Alt et al., 1980, Early et al., 1980). Sequencing analysis indicates that 92-94% of all human genes undergo alternative splicing
(Wang et al., 2008). Two to several thousands of mRNAs can be produced
from one single transcript by alternative splicing (Navaratnam et al., 1997,
Rosenblatt et al., 1997, Schmucker et al., 2000). The effects of alternative
splicing are versatile and can for example determine sex, as can be seen in
probably the most studied system of alternative splicing, the splicing of
RNA encoding the Drosophila Sex lethal protein. The RNA binding protein
Sex lethal (Sxl) is expressed specifically in female flies and represses splicing patterns that would lead to development of male flies. The repression is
due to Sxl preventing splicing factors from binding the RNA in a male splicing pattern. The Sxl transcript itself is alternatively spliced in a male or female manner, due to autoregulation (Baker, 1989). Other effects of alternative splicing can be seen in tissue-specific splicing, alternative splicing due
to cell type or alternative splicing induced by external stimulus.
The different mechanisms for alternative splicing can be categorized into
four different groups, alternative 5´ splice site selection, alternative 3´ splice
site selection, cassette-exon inclusion or skipping and finally intron retention
(Fig. 6) (Nilsen and Graveley, 2010). Alternative splicing is regulated by
splice site recognition that is controlled by the strength of the splice site, the
concentration of splicing factors and/or by the presence of splicing regulatory sequences within the pre-mRNA (Maniatis, 1991). Some splicing factors,
like SR proteins, can facilitate splice site recognition by binding to splicing
enhancer sequences and further recruit the splicing complex (Bourgeois et
al., 1999, Graveley et al., 2001). There are several different ways in which
inhibition of splice site recognition can occur. Factors binding to splicing
silencers can sterically hinder other factors from binding to splice site recognition sequences, or even enhancer sequences, preventing further recruitment
of the splicing complex. One example is hnRNP A1 binding to an intron in
HIV-1 pre-mRNA that prevents binding of U2 snRNP to a downstream 3´
splice site (Tange et al., 2001). It has also been shown that there is a coupling between transcription and splicing and that this coupling could regulate
splicing (Cheng et al., 2007, Kornblihtt, 2006, Lenasi et al., 2011, Smith et
al., 2004). The outcome of a splicing event is therefore affected by the relative ratio of activators and inhibitors, the transcription event and the speed of
elongation. Another possible influence of splicing is the secondary structure
of the pre-mRNA and more studies are needed to further examine the effect
of the secondary structure (Buratti and Baralle, 2004).
26
One type of splicing factors that are involved in alternative splicing is the
family of serine-arginine rich proteins (SR proteins) and another type is the
family of heterogeneous nuclear ribonucleoproteins (hnRNPs).
5´-splice site selection
3´-splice site selection
Cassette-exon inclusion or skipping
Intron retention
Figure 6. The different categories of alternative splicing. Boxes represent exons,
thick lines represent introns and thin lines represent alternative splicing patterns.
Adapted/modified from Nielsen et al 2010. Left panel shows pre-mRNAs, right
panel shows spliced mRNAs.
SR proteins
The family of human serine-arginine rich (SR) proteins is phylogenetically
conserved and currently contains 12 known members: SRp20, SRp30a-c,
SRp38, SRp40, SRp46, SRp54, SRp55, SRp75, 9G8 and SRSF12. These
proteins contain one or two RNA recognition motifs (RRM) at their N27
termini and a serine-arginine (RS) rich domain at their C-termini. The
RRM(s) interacts in a sequence specific manner with the pre-mRNA, although with relatively low specificity. The RS domain functions as a protein
interaction domain, especially with other RS-domain-containing splicing
factors. The nomenclature for SR proteins has recently been changed into SR
splicing factor (SRSF) and the new names are listed in table 1 (Manley and
Krainer, 2010).
Table 1. SR protein/gene names
New protein/gene name
SRSF1
SRSF2
SRSF3
SRSF4
SRSF5
SRSF6
SRSF7
SRSF8
SRSF9
SRSF10
SRSF11
SRSF12
Previous name
ASF/SF2
SC35
SRp20
SRp75
SRp40
SRp55
9G8
SRp46
SRp30c
SRp38
SRp54
SRrp35
SR proteins as regulators of splicing
SR proteins are involved in the regulation of splicing and can function both
early and late in the splicing process. In constitutive splicing it has been suggested that they participate in the exon-definition process and a number of
SR protein binding sites have been identified in constitutive exons
(Graveley, 2000). SR proteins also play a role in alternative splicing: By
binding to exonic splicing enhancers they can stimulate the usage of a 3´
splice site either by recruiting members of the splicing complex or by counteracting complexes that are inhibiting splicing (Kan and Green, 1999, Zuo
and Maniatis, 1996). SR proteins bound to upstream splicing enhancers can
stimulate the usage of the downstream 5´ splice site potentially by stimulating the binding of U1 snRNP to the splice site. SR proteins can also bind
splicing silencer elements and thereby inhibit splicing by preventing recruitment of the spliceosome. This has for instance been shown in splicing
regulation of adenovirus pre-mRNA. In this case, SR protein ASF/SF2 binds
to a splicing silencer and prevents U2AF binding to the 3´ splice site
(Kanopka et al., 1996).
Other functions of SR proteins
SR proteins participate in other RNA processing events such as targeting
mRNAs with premature stop codons to the nonsense-mediated decay path28
way. Some SR proteins shuttle between the nucleus and the cytoplasm, indicating a function also in mRNA export. In fact, SRp20, 9G8 and ASF/SF2
have been shown to be involved in nuclear export of mRNA by interacting
with TAP. Another example is the nuclear export of intronless histone H2A
mRNA that is promoted by SRp20 and 9G8 (Hargous et al., 2006, Huang et
al., 2003, Huang and Steitz, 2001, Huang et al., 2004). SR proteins can in
addition stimulate translation, e.g. IRES-mediated translation by SRp20
(Bedard et al., 2007).
Phosphorylation determines the function of SR proteins
The serines in the RS domain can be phosphorylated and the state of phosphorylation determines the function of the SR proteins (Graveley, 2000,
Kanopka et al., 1998, Xiao and Manley, 1998). The RS domain is phosphorylated by e.g. Clk/Sty kinase, cdcp34, SR protein kinase 1 (SRPK1) and
topoisomerase (Colwill et al., 1996, Mattaj, 1994, Okamoto et al., 1998,
Soret and Tazi, 2003). It has been shown that phosphorylation is needed for
efficient splice-site recognition, and that dephosphorylation is needed for
splicing catalysis (Mermoud et al., 1992, Mermoud et al., 1994). It also
seems like the dephosphorylated state more efficiently interacts with TAP
and allows the proteins to shuttle from the nucleus to the cytoplasm. In the
cytoplasm SR proteins are detached from the mRNA complex, followed by
phosphorylation of the RS domain which mediates transport back to the nucleus (Caceres et al., 1997, Kataoka et al., 1999, Lai et al., 2000, Lai and
Tarn, 2004, Swartz et al., 2007).
SRp55
One of the SR proteins, SRp55, is of particular interest in this thesis, and will
be described in some detail. SRp55, or SFRS6 (or SRp55/B52 in Drosophila)
as it is also called, is a serine-arginine rich protein with two RRMs. As for
many SR proteins, the function of SRp55 is still not fully understood. But
the protein has been reported to be involved in splicing regulation of several
different transcripts. For instance the proinflammatory protein TF where the
unspliced form is membrane bound and the spliced (promoted by SRp55) is
soluble (Tardos et al., 2008). Another splicing event regulated by SRp55 is
the calcitonin/CGRP RNA, where SRp55 regulates the splicing in a tissue
specific manner (Tran and Roesser, 2003). Splicing regulation mediated by
SRp55 has also been shown to act in an antagonistic way, competing with
other SR proteins (Chandradas et al., 2010). Other studies have reported that
the Drosophila protein SRp55/B52 targets Topoisomerase I to sites of transcription (Juge et al., 2010). SRp55/B52 has also been shown to play a role
in Drosophila cell cycle control (Rasheva et al., 2006). SRp55 (SFRS6) can
shuttle between the nucleus and the cytoplasm (Bjork et al., 2009, Sapra et
al., 2009). It has also recently been shown that SRp55 enhances translation
of unspliced HIV-1 mRNA (Swanson et al., 2010).
29
HIV-1 pre-mRNA splicing
In order to produce all the mRNAs needed for HIV-1 to be infectious, the
virus utilizes alternative splicing (Arya and Gallo, 1986, Feinberg et al.,
1986, Schwartz et al., 1990a, Schwartz et al., 1990b). HIV-1 produces one
single transcript (9 kb) that apart from functioning as genomic RNA also
encodes for the Gag and Gag-Pol proteins. This unspliced transcript can also
be spliced into mRNAs within the 4 kb class or the 2 kb class (Fig. 7). In the
9 kb transcript there are several splice sites present: four 5´ splice sites, here
referred to as splice donors (SD1-4), and nine 3´ splice sites, here referred to
as splice acceptors (SA1-4, 4a-c, 5 and 7). The splice sites can all be used in
different combinations to create more than 35 different mRNAs. The efficiencies of the splice donors depend on their complementarities to U1 snRNA while the splice acceptors are more depending on cis-regulatory elements present in the exons or the introns (O'Reilly et al., 1995). In order to
produce the correct amounts of all mRNAs, the splice acceptors in HIV-1 are
sub-optimal because of short and interrupted polypyrimidine tracts, branch
points that are not canonical and inhibitory sequences that up or down regulate the usage of splice sites (Bilodeau et al., 2001, Dyhr-Mikkelsen and
Kjems, 1995, Jacquenet et al., 2005, Marchand et al., 2002, O'Reilly et al.,
1995). This allows temporal regulation by enhancers and silencers.
HIV-1 mRNAs
The mRNAs produced from HIV-1 can be divided into three classes: 9 kb, 4
kb and 2 kb. The full-length unspliced 9 kb transcript can (i) encode for the
Gag and Gag-Pol proteins (ii) be incorporated into the virion as a genome
(iii) be spliced into 4 kb and 2 kb class of mRNAs. The 4 kb class (partially
spliced) includes the mRNAs encoding for Env, Vif, Vpr and Vpu and the 2
kb class (fully spliced) includes the mRNAs encoding for Tat, Rev Vif, Vpr
and Nef. mRNAs that are present in high abundance are env and nef,
while mRNAs like tat, vif and vpr are present in relatively low amounts.
These differences are most likely due to differences in the splicing efficiency of the respective splice site (Purcell and Martin, 1993).
30
tat
gag
vif
pol
vpr
rev
env
nef
vpu
LTR
LTR
mRNAs:
9 kb
4 kb
2 kb
Figure 7. Schematic representation of the HIV-1 genome and the different mRNA
classes. Boxes represent open reading frames, thick lines represent exons included in
the mature mRNA and thin lines represent introns removed during splicing reaction.
Circles indicate splice donors and triangles indicate splice acceptors.
Viral proteins affecting splicing
Early in the HIV-1 infection only the proteins Tat, Rev and Nef are produced
because the mRNAs encoding these proteins are the only ones completely
spliced and therefore the only ones exported to the cytoplasm. Rev is an 18
kDa protein with a nuclear localization signal. Rev goes to the nucleus and
interacts with a structured region in the env gene, the Rev response element
(RRE), present in the unspliced and partially spliced mRNAs. Rev also interacts with the cellular protein Crm1 and thereby facilitates the export of the
4 kb and the 9 kb mRNAs to the cytoplasm. mRNAs that would otherwise be
retained in the nucleus due to the presence of unused splice sites and cisacting RNA elements (Chang and Sharp, 1990, Dreyfuss et al., 2002, Rosen
et al., 1988, Schwartz et al., 1992). The export of incompletely spliced
mRNAs mediated by Rev can compete with the splicing process in a timedependent manner (Nekhai and Jeang, 2006, Pollard and Malim, 1998).
Tat has been shown to interact with the cellular protein p32, an interaction
that is increased when Tat is acetylated. Tat has additionally been shown to
interact with CDK13, a cellular factor that phosphorylates ASF/SF2, in a
complex with p32. Phosphorylation of ASF/SF2 leads to an increase in splicing of viral RNA at the expense of unspliced RNA. Acetylated Tat has been
shown to accumulate as the infection proceeds, and as a consequence this
increases the amount of unspliced HIV-1 RNA at the expense of spliced
viral RNA (Berro et al., 2006, Berro et al., 2008). Another study indicates
that Tat can regulate splicing by interacting with cellular factors in a complex that recruits SR proteins AFS/SF2 and SC35 to the sequence in exon 5
called guanosine-adenosine-rich (GAR). This increases the expression of env
mRNA (Jablonski et al., 2010).
31
Silencers and enhancers on the HIV-1 pre-mRNA
Several elements have been found (in vitro and in vivo) to function as enhancers or silencers for HIV-1 pre-mRNA splicing. Some of those will be
described in the following sections (Tazi et al., 2010). The splice sites are
indicated in Fig. 8.
tat
gag
vif
pol
vpr
rev
env
nef
vpu
LTR
SD1
SD2 SD3
SA2 SA3
SD5
SA5
SA4b
SA4a
SA4c
SA4
SA7
LTR
Figure 8. The HIV-1 splice sites. Boxes represent open reading frames, circles indicate splice donors and triangles indicate splice acceptors. Numbers of splice donors
(SD) and splice acceptors (SA) are indicated.
Splice acceptor 2 and splice donor 2
Splice acceptor 2 is regulated by a splicing enhancer within exon 2 that facilitates inclusion of exon 2, producing vif mRNA. This enhancer binds to
SRp75. A splicing silencer controlling splice donor 2 is positioned downstream of exon 2 and down regulates the expression of vif mRNA (Exline et
al., 2008).
Splice acceptor 3 and splice donor 3
Splicing into splice acceptor 3 can result in vpr mRNA. A silencer positioned within exon 3 is called ESSV and inhibits the vpr splice acceptor
SA3. This is due to hnRNP A/B binding to the silencer and antagonizing
binding of U2AF65 to the viral polypyrimidine tract (Bilodeau et al., 2001,
Domsic et al., 2003). It has also been reported that splicing into SA3 is activated by ASF/SF2 and that this is in competition with hnRNP A/B (Ropers
et al., 2004, Zhu et al., 2001). In our paper from 2010 (Paper I) we show that
SRp55 inhibits SD3 by interacting with an RNA element in exon 5 (Tranell
et al., 2010).
Splice acceptor 4
Splicing into SA4 will result in tat mRNA production. Within exon 4 there
are two silencers, ESS2 and ESS2p, both inhibiting splicing into exon 4 and
32
thereby the production of tat mRNA – the ESS2 by binding hnRNP A1 and
the ESSp2 by binding hnRNP H (Amendt et al., 1994, Jacquenet et al.,
2001). An enhancer within exon 4 has been named exonic splicing enhancer
2 (ESE2) because it attenuates ESS2 and thereby enhances splicing into
SA4. This enhancer interacts with the SR protein SC35 (Zahler et al., 2004).
Splice acceptor 5 and splice donor 5
Another enhancer, named GAR, is positioned within exon 5 and has a positive effect on the usage of SA5, the nef splice acceptor, and SD5, used to
produce all 2 kb mRNAs. GAR interacts with SRp40 and ASF/SF2 (Caputi
et al., 2004, Kammler et al., 2001). Our results (Paper I) show that SRp55
interacts with GAR and inhibits SD3 (Tranell et al., 2010).
Splice acceptor 7
SA7 is used to produce all mRNAs in the 2 kb class. An intronic silencer
(ISS) found to inhibit SA7 is located within the intron upstream of SA7
(Tange et al., 2001). The exonic splicing silencer ESS3 suppresses SA7 by
blocking U2 snRNP. Both ISS and ESS3 interact with hnRNP A1 (Amendt
et al., 1995). Close to ESS3 within exon 7 there is an enhancer element
called ESE3 that activates SA7 that can also function as a silencer when
binding to ASF/SF2 (Staffa and Cochrane, 1995, Zhu et al., 2001).
SR proteins and HIV-1 pre-mRNA splicing
SR proteins have been found to affect HIV-1 pre-mRNA splicing: SC35,
9G8 and SRp40 increase the amounts of tat mRNA. SC35 and SRp40 by
binding to sequences in exon 4, which probably counteract the splicing inhibitory hnRNP A1 binding to the pre-mRNA (Hallay et al., 2006, Ropers et
al., 2004). siRNA knockdowns of SR proteins SRp40 and SC35 in fact decreased the level of tat mRNA (Jablonski and Caputi, 2009). ASF/SF2 has
been found to up-regulate the HIV-1 vpr mRNA and to increase splicing at
SA2 and SA3 (Jacquenet et al., 2005, Ropers et al., 2004). The levels of Nef
protein have been shown to decrease upon over expression of SC35, SRp40
and ASF/SF2 (Ropers et al., 2004).
SR proteins and HIV-1 mRNA biogenesis
Apart from the involvement in splicing of HIV-1 pre-mRNA, SR proteins
have been reported to have other functions in HIV-1 mRNA biogenesis. For
instance in our paper from 2011 (Paper II) there are indications that SR proteins can stabilize and increase nuclear export of viral mRNAs (Tranell et
al., 2011). Another possible function for SR proteins is to facilitate translation of the viral unspliced mRNA encoding Gag and Gag-Pol protein.
Unspliced mRNAs are not associated with exon junction complexes that are
needed for an mRNA to be translated. Translation of the unspliced gag
33
mRNA must therefore be regulated in a different manner. This regulation is
still unknown but there are reports indicating this could take place with the
help of SR proteins (Swanson et al., 2010).
Antiviral therapy and HIV-1 splicing
A new approach to fight HIV-1 infection could be to interfere with the essential splicing, either by targeting a splice site or by targeting splicing factors. Researchers have used antisense molecules against HIV-1 splicing elements. One example is the antisense sequences targeted against splice donor
5 and the GAR sequences, which have been shown to induce exon skipping
and thereby inhibit tat and rev mRNA splicing (Asparuhova et al., 2007).
Another way of interfering is to target splicing factors, indirectly or directly.
An indirect way would be to target factors that regulate splicing proteins e.g.
kinases that phosphorylates SR proteins or using modified U1 snRNA to up
or down regulate the usage of specific HIV-1 splice sites. A more direct way
has been studied using small molecules called indole derivatives that can
target SR proteins (Bakkour et al., 2007, Mandal et al., 2010, Soret et al.,
2005, Wong et al., 2011).
Indole derivatives
Indole derivatives are small molecules that can interact with SR proteins and
alter or inhibit their function in pre-mRNA splicing regulation. The most
plausible mechanism to explain how they interfere with the splicing regulation is by changing the phosphorylation status of the SR proteins. Indole
derivatives are potent inhibitors or re-directors of HIV-1 RNA production in
chronically infected cells where they interact with SR proteins. One specific
indole derivative called IDC16 was reported to block HIV-1 viral production
in infected PBMCs or macrophages by interacting with ASF/SF2 (Bakkour
et al., 2007, Fukuhara et al., 2006, Soret et al., 2005).
34
The present investigation
Aims of the study
In order to produce all the mRNAs needed for HIV-1 to be infectious the
virus uses alternative splicing. From one single transcript more than 35 differently spliced mRNAs can be produced. This alternative splicing is highly
regulated in order to generate the correct amount of each mRNA. Therefore
more and more research is being done within this field. Splicing factors or
cis-sequences within the HIV-1 RNA affecting the splicing pattern in HIV-1
are of great interest when designing new potential antiviral agents. We have
studied the splicing regulation in the hope that this knowledge could be used
to design new antiviral treatments. The first step in gaining this understanding is to identify regulatory elements or factors that are important for HIV-1
pre-mRNA splicing. We have identified cis-elements in the HIV-1 genome
that are required for HIV-1 splicing regulation. We have also identified cellular factors that regulate HIV-1 mRNA splicing and we have studied how
these factors affect the HIV-1 splicing pattern.
Experimental system
To study regulation of HIV-1 pre-mRNA splicing we used the sub-genomic
HIV-1 plasmid pDP (Fig. 9), constructed in our lab. This plasmid contains
the entire HIV-1 genome with the exception of a 2 kb deletion within the pol
gene, which renders the genome non-infectious. The natural HIV-1 LTR
promoter drives the transcription from this plasmid. We also used subgenomic plasmids and cDNAs with different deletions and mutations to find
the regions of importance for HIV-1 pre-mRNA splicing regulation.
For studies in cells the plasmids were transfected into cervical cancer
cells (HeLa) and the splicing pattern was analyzed by northern blot analysis
or RT-PCR. To identify factors that are functionally involved in HIV-1 premRNA splicing, plasmids expressing the factors of interest were overexpressed in co-transfection experiments with sub-genomic HIV-1 plasmids
in HeLa cells. For studies in cell lines containing integrated HIV-1 genome
(HLfB cells) SR protein expression plasmids were transfected into these
cells. The amount of HIV-1 p24gag protein in cell culture media was determined by p24gag antigen capture ELISA.
35
tat
gag
vif
pol
vpr
rev
env
nef
vpu
LTR
LTR
mRNAs:
9 kb
4 kb
2 kb
Figure 9. The HIV-1 sub-genomic plasmid pDP, containing a 2 kb deletion in the
pol gene, and the mRNA classes it produces. Boxes represent open reading frames,
triangles represent splice acceptors and circles represent splice donors. Thick lines
represent exons and thin lines represent spliced introns.
Results
Paper I
Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5'-Splice site of
Exon 3
HIV-1 uses alternative splicing in order to produce all the mRNAs that are
needed for the virus to be infectious. From one single transcript more than
35 differently spliced mRNAs are produced. Alternative splicing is highly
regulated and in this study we tested the effect of the splicing factors SR
proteins on HIV-1 pre-mRNA splicing. To study this we used sub-genomic
HIV-1 expression plasmids. The plasmids were transfected into HeLa cells
in the absence or presence of SR protein expression plasmids. Cytoplasmic
RNA was extracted 24 hours post-transfection and the splicing pattern of
HIV-1 was analyzed with northern blotting and RT-PCR.
The findings in this paper showed that the intracellular concentration of
SR proteins is of great importance for HIV-1 to be able to produce the correct amounts of mRNAs. Variations in concentrations of SR proteins lead to
great changes in HIV-1 pre-mRNA splicing pattern.
HIV-1 vpr mRNA 13a7 is a partially spliced mRNA because it contains
an intron between exon 3 and exon 4. Incompletely spliced mRNAs are
normally trapped in the nucleus where they are degraded. This would happen
to the gag-pol and env mRNAs unless Rev would bind to RRE and aid in the
36
export of these mRNAs. In contrast, Rev does not bind to the vpr mRNA
13a7 that also contains an intron and it is therefore unclear how these
mRNAs are made and how they reach the cytoplasm (Fig. 10). We found
that SRp55 and SRp75 induced the production of HIV-1 tat mRNAs 147 and
14E as well as vpr mRNA 13a7.
tat
gag
vif
pol
vpr
rev
env
nef
vpu
LTR
LTR
RRE
RRE
9 kb
4 kb
2 kb
vpr 13a7 mRNA
Figure 10. Indication of Rev response element (RRE). The HIV-1 genome at the top.
Boxes represent open reading frames, circles indicate splice donors and triangles
indicate splice acceptors. Thick lines represent exons present in mRNA and thin
lines represent spliced introns. The RRE is indicated on the 9 kb and the 4 kb
mRNA classes. The 2 kb class and the vpr 13a7 mRNA does not contain the RRE
and is therefore not exported to the cytoplasm with the help of Rev.
We wanted to study further the SRp55 regulation of vpr mRNA. By introducing mutations in the HIV-1 genome, we found that induction of vpr
mRNA due to over expression of SRp55 was dependent on a previously
identified sequence in exon 5 named GAR. We could also show that SRp55
interacts directly with the GAR RNA. The interaction inhibited the function
of GAR as a splicing enhancer. More specifically, SRp55 inhibited SD3 in
exon 3 and thereby induced vpr mRNA 13a7. We also concluded that SRp55
interaction with GAR is in competition with SRp40 and that the production
of vpr mRNA is determined by the relative concentrations of SRp40 versus
SRp55 (or SRp75). This competition is more complex than simply competing for binding because inhibition of SD3 requires not only the RRM of
SRp55 but also the RS domain. This indicates that inhibition requires SRp55
interaction with an additional, still unidentified, factor.
37
Paper II
Inhibition of Splicing by Serine-arginine Rich Protein 55 (SRp55) Causes the Appearance of Partially Spliced HIV-1 mRNAs in the Cytoplasm
In paper I we showed that SRp55 inhibits SD3 and thereby induces the production of 13a7 vpr mRNA. HIV-1 Vpr protein is incorporated into the virion and into the viral pre-integration complex. The functions of Vpr are diverse and it has been shown to stimulate transcription from the HIV-1 LTR
promoter, affect the reverse transcription process, cause G2 cell cycle arrest,
induce apoptosis and suppress immune response. These mechanisms require
production of vpr both early and late in the viral life cycle. We therefore
wanted to further study the regulation of HIV-1 mRNA processing by
SRp55.
According to our results the majority of mRNAs in the nucleus produced
from 13a7 cDNA plasmid pNL13a7 were unspliced 13a7 mRNA and a minority was the fully spliced 1357 mRNA. This showed that unspliced 13a7
mRNA was trapped in the nucleus. We have previously shown that SRp55
increases the production of 13a7 vpr mRNA by inhibiting SD3. And in this
paper we concluded that this inhibition caused an increase in the amount of
unspliced 13a7 vpr mRNA in the cytoplasm, due to an increased nuclear
export of this mRNA.
Further on, we wanted to see if SRp55 could also induce the production
of partially spliced 4 kb mRNAs that are dependent on the Rev protein and
produced late in the viral life cycle, and more specifically vpr mRNA 13aE.
We showed that SRp55 inhibited splicing of viral mRNAs spliced at SD5
suggesting that SRp55 inhibits SD5. This inhibition resulted in the appearance of partially spliced mRNAs vpu, env and vpr (13aE) in the cytoplasm.
The increase in cytoplasmic amounts of these mRNAs also indicated an increase in mRNA nuclear export in the presence of SRp55.
The viral protein Rev is produced from a fully spliced mRNA and the
protein associates with partially spliced and unspliced viral mRNAs and
facilitates their nuclear export. Because intron-containing mRNAs are considered immature, these mRNAs survive with Rev, where they would otherwise be trapped in the nucleus and degraded. We studied whether SRp55 and
SRp75 could increase the amount of partially spliced mRNAs in the cytoplasm of a cell line containing integrated copies of a Rev mutant virus and
therefore is unable to produce infectious virus particles. The results showed
that transfection of SRp55 and SRp75 into these cells increased the amount
of p24-Gag, a viral protein present in HIV-1 virions and in the cell culture
media. This protein is produced from an unspliced mRNA. The increase of
p24-Gag was nevertheless as great as in Rev mediated export of mRNA.
38
The results from the transfection experiment together with the Rev mutant
virus experiments suggested that high levels of SRp55 can replace the function of Rev in an early stage of infection, before the production of Rev has
reached higher levels. It has previously been reported that Vpr has an effect
on transcription of HIV-1 genome and that Vpr can suppress the immune
system during an HIV-1 infection. High levels of SRp55 during early infection will lead to early production of Vpr and more efficient transcription.
Vpr could therefore have important functions initially during an HIV-1 infection.
The increased nuclear export of vpr mRNA as could be seen during over
expression of SRp55 could be due to two things: on the one hand SRp55
could mediate transport by binding mRNA and nuclear pore complex, on the
other hand SRp55 could interfere with the interaction between partially
spliced mRNA and splicing factors and thereby release vpr mRNA for export. Some SR proteins have previously been shown to interact with nuclear
export factor TAP/Nxf1. Some, e.g. SRp55, can shuttle between the nucleus
and the cytoplasm.
Paper III
Indole-related Compounds from the National Cancer Institute (USA)
Repository do not Inhibit HIV-1 Replication in Primary Blood Mononuclear Cells
Indole derived substances have previously been shown to inhibit HIV-1 replication. We tested the effect of indole-related substances on HIV-1 gene
expression in transient transfections and in primary cell cultures infected
with HIV-1. For transfection assays we used a sub-genomic HIV-1 expression plasmid called pDP. This plasmid was transfected into HeLa cells in the
absence or presence of 10 µM of each compound. The levels of HIV-1
mRNAs in cytoplasmic RNA extracts 24 hours post-transfection were determined by northern blot. HIV-1 gag protein levels were determined by
western blot with proteins extracted 24 hours post-transfections.
In our study some of these substances had an inhibitory effect on HIV-1
gene expression in cells transiently transfected with the subgenomic plasmid
pDP, which could be seen on northern blotting and western immunoblotting.
For studies on HIV-1 infected cells we used primary peripheral blood mononuclear cells (PBMCs). To measure virus particles, the levels of extra cellular HIV-1 p24 gag protein were used in ELISA. The results showed that
some of the substances were toxic to the cells, and were therefore not tested
further. None of the substances tested inhibited replication of HIV-1 in primary cells. Although the promising results obtained in transfection studies,
inhibition of HIV-1 gene expression was not seen in infected cells. These
39
results demonstrated important differences between transfection assays performed in cell lines and infection assays in primary cells.
40
Concluding remarks and future prospects
At the end of 2010, 34 million people were living with HIV-1 infection.
HIV-1 is the causative agent of AIDS, which causes millions of deaths every
year. There are several antivirals against HIV but due to the virus high mutability, causing resistance, there is a constant need for new antivirals. In
order to develop new strategies of treatment and prevention, we need to
know more about the virus and the way it behaves in our cells.
The splicing process of HIV-1 pre-mRNAs is highly regulated and is essential for the virus to be infectious. This process is a potential target in development of new treatments. It is therefore of great interest to understand
how the splicing process is regulated for HIV-1 pre-mRNAs. In this thesis I
have discussed the regulation of HIV-1 pre-mRNA splicing. We identify
regulatory elements associated with splicing within the viral genome as well
as cellular splicing factors that affect the HIV-1 pre-mRNA splicing pattern.
More specifically, we shed some light on how the HIV-1 vpr mRNA is
regulated. The cellular protein SRp55 and SRp75 regulate the splicing of
HIV-1 pre-mRNA by inhibiting splice donor 3 (SD3), leading to induction
of HIV-1 vpr mRNA. We also discover that the inhibition caused by SRp55
is due to its interaction with an RNA element called GAR, located in the
viral exon 5. According to our results the regulation of vpr mRNA is dependent on the relative concentrations of different SR proteins. We also
show specifically that there is competition between SRp40 and SRp55 in the
regulation of tat versus vpr mRNA, due to competitive interaction with GAR
between these two SR proteins. This competition is not just due to steric
hindrance, but requires other factors that are still unknown. It would be interesting to further study the mechanism of SRp55 splicing inhibition of
SD3. What other factors are required for this inhibition? This could for instance be tested in vitro with immunoprecipitation by mixing vpr mRNA
with tagged SRp55 and adding nuclear extract and also by fishing up tagged
SRp55 and analyzing binding partners with mass spectrometry.
We find that inhibition of SD3 by SRp55 not only leads to induction of
vpr mRNA but also to an increased export of vpr mRNA to the cytoplasm.
The vpr mRNA contains an intron and would therefore be trapped in the
nucleus and degraded. Somehow the vpr mRNA can overcome this and be
exported to the cytoplasm for translation. The viral protein Rev facilitates
export of unspliced and partially spliced viral mRNAs in the 9 kb and 4 kb
class by interacting with RRE, an RNA element present on these mRNAs.
41
Vpr mRNA does not contain the RRE and can therefore not be exported with
the assistance of Rev. We therefore speculate that SRp55 not only inhibits
the SD3 to increase the production of vpr mRNA, but also that it can aid in
the nuclear export of this mRNA. Another interesting question is therefore
how SRp55 can facilitate the export of vpr mRNA to the cytoplasm. What
mechanism is required for this export and is SRp55 associated with vpr
mRNA in the cytoplasm? Is SRp55 mediating export by binding mRNA and
nuclear pore complex or some other export factor, or is it interfering with the
interaction between partially spliced mRNA and splicing factors thereby
releasing export inhibitory factors from the mRNA? Some SR proteins have
been shown to enhance translation. It would be interesting to see if SRp55
could, not only induce production and export of vpr mRNA, but also enhance the translation of this mRNA.
Our results show that SRp55 can have several functions in the regulation
of HIV-1 splicing: by inhibiting a splice donor, and by facilitating the export
of an incompletely spliced mRNA to the cytoplasm. It would be interesting
to study the levels of SRp55 in the cell during HIV-1 infection. One could
speculate that the levels would change during infection. It would also be
interesting to see how the HIV-1 splicing pattern changes due to changes in
SRp55 levels in infected cells. What happens during infection if SRp55 is
knocked out? And how is the cell affected by this knock out? If SRp55 becomes a potential drug target it should preferably not be essential for the
cell. One could generate a knock out mouse to observe the effects of SRp55
knock out.
Vpr is essential during viral infection and has several functions. It facilitates infection by mediating the nuclear import of HIV-1 pre-integration
complex in non-dividing cells, it induces cell cycle arrest in the G2 phase, it
transactivates the viral promoter and it induces cell death of immune cells
(Agostini et al., 1996, Felzien et al., 1998, He et al., 1995, Kogan and
Rappaport, 2011, Le Rouzic et al., 2002, Nie et al., 1998, Roshal et al.,
2001). One possible antiviral strategy would be to interfere with the interaction between SRp55 and GAR, which could prevent vpr mRNA production.
By using antisense oligos towards GAR, the interaction between SRp55 and
GAR may be blocked. These antisense oligos would have to be specific for
GAR and should preferably not interact with genomic sequences. In fact,
antisense strategies targeting exon 5 have been tested with exon skipping and
decreased viral production as a result (Asparuhova et al., 2007). Another
approach could be to use small molecules that interact with SRp55. Small
molecules called indole derivatives can interact with SR proteins and alter or
inhibit their function in pre-mRNA splicing regulation. These molecules are
potent inhibitors or re-directors of HIV-1 RNA production (Bakkour et al.,
2007, Fukuhara et al., 2006, Soret et al., 2005). We test the effect of indolerelated substances on HIV-1 gene expression in transient transfections and in
primary cell cultures infected with HIV-1. Unfortunately none of these sub42
stances have any effect on HIV-1 replication. One reason is that the substances are toxic to the cells. To study this further we would probably use a
more efficient screening method initially, where more substances could be
tested at the same time.
Because splicing is essential for HIV-1 to be infectious it is important to
find cis-elements and transacting factors that are regulating this splicing
process. Both in vitro and in vivo methods are used when searching for these. The in vitro methods are fast and make it possible to study a specific
RNA element without disturbing sequences that surround it. However, the
splicing process is very complex. Interaction of a protein upstream of the
sequence of interest could affect the interaction to the actual sequence. The
secondary structure of the RNA could also be important in splicing regulation (Buratti and Baralle, 2004). In vivo experiments with full-length premRNAs should give a better picture of the splicing due to the secondary
structure. Taken together both in vitro and in vivo (transfections as well as
infections) studies are important in the search for splicing regulatory elements or factors in HIV-1 pre-mRNA splicing.
43
Acknowledgements
Vägen till denna avhandling har varit ”long and winding”. Men som Gunde
Svan brukar säga ”Går man den väg alla andra går, kommer man inte längre
än någon annan”. Jag skulle vilja tacka alla som hjälpt mig:
Min handledare Stefan Schwartz för att jag fick möjligheten att forska i ditt
lab! Du har alltid varit tillgänglig och jag har vågat ställa de mest korkade
frågorna utan att känna mig dum.
Min bihandledare Göran A kusjärvi för alla råd och tips under denna tid.
Du ger virusforskningen på IMBIM en stabilitet som är oerhört värdefull.
Catharina Svensson, för att du delat med dig av all din kunskap. Du är en
inspirationskälla.
Min examinator Göran Magnusson för alla intressanta anekdoter på fredagsmötena.
Energiska och emotionella Monika Somberg, för att citera dig själv:
”partner in crime”. Vi visste inte vad som väntade när vi ungefär samtidigt
började denna resa. Det har varit både upp och ner och hit och dit. Tack för
alla hjälp och för alla skratt (och tårar) som du gett mig! Man blir aldrig
uttråkad eller förvånad när man hänger med dig! (Och tack för alla lösningar
jag ”fått låna”.)
Jag är oerhört glad över att jag har fått lära känna alla splicegirls: Susanne
för ditt lugn och din allt-ordnar-sig-inställning. Och ett oerhört stort tack för
att du bidrog med experiment. Heidi för att du alltid är positiv och hjälpsam.
Ellenor för dina smarta och satiriska kommentarer. Cecilia för din uppriktighet och dina otroliga labkunskaper. Sara för din empati och ditt härliga
skratt. X iaoze för att du gett mig en inblick i den kinesiska världen. Lena för
att du höll ordning på det mesta. Sofia du var i labbet en kvart – men det var
roligt. Tack också till Ning, A nette, Samir och Hilary. Och tack till de tidigare medlemmarna i gruppen, X iaomin och Margaret, för att ni introducerade mig till labbet och lärde mig grundteknikerna.
Barbro, Erika, Kerstin, Marianne, Olav och Rehné. Utan er skulle IMBIM
inte vara någonting.
Eva Maria Fenyö och Elzbieta V incic för all hjälp på P3-labbet i Lund.
DA -gruppen för roliga lunchdiskussioner. Det får alltid plats några till vid
ett runt bord.
Sara Brunt och Christer Harbeck för att ni gett mig redskap för resten av
livet.
44
A nu Seensalu för din uppmuntran och stora förståelse och för att du lät
mig ta tjänstledigt för att slutföra detta. Tack också till alla kollegor på Natur
& Kultur, den goda stämningen på jobbet har gett mig energi till detta.
Biologfamiljen: Hanna S för alla klantigheter och alla roliga studentupplevelser. ”Man kan alltid tvätta händerna i lite NaOH”. Elisabet för att du
alltid är ärlig och rak. Karin för roliga (och jobbiga) månader i Indien. Emma
för din humor som alltid lockar fram skratt. Hanna B för att du är envis och
smart. Tillsammans med alla andra medlemmar i biologifamiljen har ni gjort
tiden i Uppsala till en av de bästa!
Julitatjejerna – vi har känt varandra sen vi var yngel, tack för att ni fortfarande är mina vänner! Maria för att du alltid finns där och för att du är lite
galen. Elin för de roliga resorna tillsammans. Hanna för att du ger mig pysselinspiration. Jill för de filosofiska stunderna.
Jonas familj för att ni tagit emot mig med öppna armar. Ni är så härliga
allihop!
Mina kära föräldrar. Tack mamma och pappa för all kärlek och allt stöd ni
alltid har gett mig. Tack för att ni fyllt min uppväxt med trygghet och böcker, ”En barndom utan böcker, det vore ingen barndom”.
Allrakäraste systrar, ni är världens bästa och jag är så stolt över er! Erika,
tack för din humor och för att du gör mig så glad! Och tack för att du fyller
på mitt bibliotek av Astrid-filmer. Du är bäst! Kristin, min smarta, idésprutande syster som kan allt. Vad skulle jag göra utan dig?! Tack för alla fina
kläder du syr åt mig. Du kan bli allt du vill!
Ett stort tack också till morfar och mormor, farfar (var du än är) och farmor, A dolf.
Min älskade Jonas, du är stommen i mitt liv. Din kärlek och ditt stöd är
ovärderligt. Tack för att du ”plogat” åt mig – utan din hjälp hade skrivandet
tagit mycket längre tid, och vårt hem sett ut som ett bombnedslag. Nu när
även jag är färdig kan vi tillsammans gå vidare mot allt härligt som väntar
oss! ”…så länge det finns stjärnor över oss…”
45
46
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