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Copyright: © 2015 Weining Han, et al.
Explore and Exploit
Research Article
Journal of AIDS and Immune Research
Open Access
Forced Complementation between Subgenomic RNAs: Does Human
Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral
Core, Cytoplasm, or Early Endosome?
Weining Han1, Yuejin Li2, Bernard S. Bagaya3, Meijuan Tian2, Mastooreh Chamanian3, Chuanwu Zhu4, Jie Shen1 and Yong Gao2,3*
1
Suzhou Center for Disease Control and Prevention, Suzhou, China
Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave, Cleveland, Ohio 44106, USA
3
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA
4
The Fifth People’s Hospital, Suzhou, China
2
Received Date: August 08, 2014, Accepted Date: February 24, 2015, Published Date: March 02, 2015.
*Corresponding author: Yong Gao, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave, Cleveland,
Ohio 44106, USA, E-mail: [email protected]
Abstract
Although the process of reverse transcription is well elucidated, it
remains unclear if viral core disruption provides a more cellular or viral
milieu for HIV-1 reverse transcription. We have devised a method to
require mixing of viral cores or core constituents to produce infectious
progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which
HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each
other and when together can complete viral reverse transcription.
Only the heterodiploid virus containing both the nfl and cplt_R/U5/
gag/Δpol sgRNAs can complete reverse transcription and propagate
infectious virus upon de novo infection. Dual exposure of U87.CD4.
CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/
Δpol virus particles did not result in productive virus infection. On the
other hand, in early endosomes, the HIV-1 sgRNAs released from viral
cores can retain function and complete the reverse transcription and
result in productive infection. These findings confirm the assumptions
that, in natural infection, HIV-1 cores, and likely other retrovirus cores,
remain largely intact and do not mix/fuse in the cytoplasm during the
reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA
(produced through transfection) could not help the complementary
sgRNA in the viral core to complement the reverse transcription process.
Keywords: Subgenomic RNAs; Human Immunodeficiency Type 1
Virus; Reverse transcription
Introduction
HIV-1 enters target cell via specific binding of viral envelope
glycoprotein gp120 with cell receptors CD4 and CCR5 (or CXCR4)
[1]. Entry can occur on the cell membrane with or without
endocytosis. Reverse transcriptase is triggered upon expulsion
of the core into the cytoplasm after viral-host membrane fusion.
Studies suggest that the core is partly disrupted to permit influx
of dNTPs, to promote reverse transcription, and to eventually form
the pre-integration complex which is actively transported to the
nucleus through a nuclear pore [2-4].
The key reverse transcription (RT) event usually lasts for
8-12h and is a very complicated and highly ordered process. In
addition to viral reverse transcriptase, the process may involve
the participation of cellular elements such as the cytoskeleton
[5] and other viral proteins, such as Tat and Vif [6]. Although the
process of reverse transcription is well characterized, the role of
the viral capsid during reverse transcription remains unclear [7,8].
Previous studies [9,10] showed that uncoating occurs shortly
after the membrane fusion event. This uncoating is necessary for
the formation of the reverse transcription complex (RTC) [11-14].
However, the conical core structure is unstable and very sensitive
to even the mildest detergent treatment used in these density
J Aids Imm Res
gradient analyses [15], thus, it is quite possible that the loose
structure observed in purified cores does not reflect a more minimal
dissociation of their components in the cytoplasm after virus entry
and early reverse transcription events. Direct observation by
scanning electron microscopy (SEM) indicates that uncoating does
not occur as an immediate post-fusion event, but rather during
late synthesis of proviral DNA synthesis and transport of the preintegration complex (PIC) to the nuclear pore [16]. In support of
this hypothesis, we discovered that siRNAs associated with the
RISC complex could not degrade HIV-1 genomic RNA following
virus entry suggesting inability to penetrate the core. However, core
dissolution was sufficiently increased by the addition of rhesus
TRIM5α such that the siRNA-RISC complex could then degrade
genomic RNA soon after virus entry [17]. This study again suggests
that, even though the viral core possibly becomes loose to facilitate
viral reverse transcription, but is still able to prevent the access of
big host molecules in the host cells.
Reverse transcription is initiated from the host tRNALys3, acting
as a primer, and annealed to the primer binding site (PBS) located
downstream of the 5′-LTR of the HIV-1 RNA genome. The first strong
stop of (-) strand DNA synthesis occurs following transcription
of the U5 and R (repeat) regions, which then triggers RNase H
degradation of this RNA template, freeing the (-) strand strong
stop DNA to pair with the R region on the opposite end of the same
genomic RNA template (intrastrand) or the other genomic RNA
template (interstrand) within the core. The preference for an intraversus inter-strand switch has been debated for over 30 years. Our
recent study had separated the HIV-1 genome into two distinct RNA
species, the first subgenomic lacking the 5’ R and U5 regions (i.e. near
full length or nfl sgRNA) and second only containing 5’ R, U5, PBS,
and necessary RNA packaging elements (i.e. complementing or cplt
RNA). The nfl sgRNA can also serve as mRNA to translate the entire
HIV-1 proteome in the correct stoichiometry, which subsequently
results in virus particle production. However, only heterodiploid
HIV-1 particles containing both the nfl and cplt sgRNAs can complete
reverse transcription upon de novo infection (Figure 1) [18]. It is
important to note that the cplt sgRNA only acts as a template for (-)
strand strong-stop DNA synthesis which can then jump onto to the
nfl sgRNA to complete proviral DNA synthesis and virus propagation.
This bipartite genome system provided a unique method to explore
HIV-1 core stability and possible core mixing/fusing within the
cytoplasm via dual infections or transfection with virus particles
harboring only one or the other sgRNAs. We could also express
these sgRNAs in the cytoplasm and look for diffusion into virus
core. Possible core mixing was assessed by completion of reverse
transcription and virus propagation. Theoretically, if the substantial
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Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
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Figure 1: HIV-1 production system with two complementary HIV-1 RNA genomes. (A) Two plasmids, pREC_nfl_NL4-3, which transcribes near full length of
HIV-1 RNA (lack of 5′LTR) and produces the full cadre of HIV-1 proteins, and pREC_cplt_R/U5/gag/Δpol, which contains R, U5, PBS, packaging sequence, gag,
and partial pol genes. (B) Virus from co-transfection of 293T cells containing cplt_R/U5/gag/Δpol and nfl HIV-1 sgRNAs can complete the reverse transcription
process in a manner analogous to the intrastrand model of retroviral reverse transcription.
core dissociation is required for HIV reverse transcription, then two
HIV-1 cores in the cells or free sgRNAs may mix to mediate complete
reverse transcription and lead to virus propagation. Core mixing/
fusing could even be promoted by the notion that HIV-1 core are
transported to the microtubule complex and subsequent migrated
along tubulin to the nuclear pore [19]. However, if core structure
is maintained and cores cannot mix during virus entry and early
reverse transcription events, then these two virus particles would
be incapable of completing reverse transcription and mediating a
productive infection. In relation to this hypothesis, we set up several
different conditions for co-exposing cells to the virus particle
containing the nfl and/or cplt sgRNAs in association to high levels of
mixing of these subgenomic RNAs.
Methods
Plasmids
The plasmids pREC_nfl_NL4-3 (containing all of the HIV-1
sequences except the R and U5 sequences) and pREC_cplt_R/U5/
gag/Δpol (containing R, U5, PBS, and gag and partial pol sequences)
were constructed in our lab [18]. The packaging plasmid ∆R8.91
(encoding HIV gag and pol proteins), plasmid pREC_HIV env
(encoding HIV-1 envelope glycoprotein), and plasmid pMD.G
(encoding vesicular stomatitis virus (VSV) G envelope glycoprotein),
were kindly provided by Dr. Stanton L. Gerson [20].
Cell culture
The U87.CD4.CXCR4 cell line(human glioma cell line expressing
CD4 and CXCR4 receptors) was obtained from the AIDS Research
and Reference Reagent Program and maintained in Dulbecco’s
modified Eagle medium (DMEM) with 15% FBS supplemented
with 100U of penicillin, 100mg of streptomycin, 300μg of G418
(Life Technologies, Inc.), and 1μg of puromycin per ml. 293T cells
were obtained from the American Type Culture Collection and
were grown in DMEM with 10% FBS supplemented with 100U of
penicillin and 100μg of streptomycin per ml. Both cell lines were
grown at 37°C in 5% CO2.
Pseudotyped virus production and virtual TCID50
Pseudotyped viruses were produced as described by Zielske
J Aids Imm Res
et al [20]. Using Lipofectamine 2000 , 293T cells were triple
transfected with pREC_cplt_R/U5/gag/Δpol (or pREC_nfl_NL43), pSM-WT (or pMD.G), and ∆R8.91 at a mass ratio of 3:1:3 to
produce: pseudoviruses containing HIV-1 cplt_R/U5/gag/Δpol
sgRNA wrapped with HIV-1 envelope (Virus #1) or VSV-G envelope
(Virus #2), pseudoviruses containing HIV-1 nfl sgRNA with HIV-1
envelope (Virus #3), pseudoviruses containing HIV-1 nfl sgRNA
with both HIV-1 and VSV-G envelopes (Virus #4), and pseudoviruses
containing both sgRNAs wrapped with HIV-1 envelope (Virus #5)
(Figure 2). Forty-eight hours after transfection, virus-containing
cell-free supernatants were collected and stored at -80°C for
further use.
Produced pseudotyped viruses were titrated via virtual TCID50
assay as previously described [21]. Briefly, the sampled viruses
were prepared using serial four-fold dilutions, along with one
reference virus with a known TCID50. 10μl of each dilution of
each virus was transferred to the round bottom 96-well plate (in
triplicate) for the subsequent RT assay. The obtained RT value of
the reference virus was plotted versus infectious units (IU) at each
dilution in the linear range, and an equation was generated based
on this relationship. The virtual TCID50 of the sample virus was
calculated by inserting the RT value into the equation.
Transfection and infection
Twenty four hours after being plated (3×104 cells per well in
48-well plates), the U87.CD4.CXCR4 cells were transfected with
certain plasmids, i.e. pREC_cplt_R/U5/gag/Δpol or pREC_nfl_NL43. 24 hours post-transfection, the cells were exposed to different
pseudoviruses to create different settings for viral sgRNAs
(i.e. in the cytoplasm and/or viral core through different entry
pathways). For entrance through the HIV envelope pathway, U87.
CD4.CXCR4 cells were transfected with pREC_cplt_R/U5/gag/
Δpol and infected with Virus #3, transfected with pREC_nfl_NL4-3
and infected with Virus #1, or infected with virus #5 only (Figure
5A). For entrance through the VSV-G envelope pathway, U87.CD4.
CXCR4 cells were transfected with pREC_cplt_R/U5/gag/Δpol and
infected with Virus #4, or transfected with pREC_nfl_NL4-3 and
infected with Virus #2 (Figure 6A). Viral infectivity was monitored
at different time points by RT assay post-infection. Co-infections
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Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
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Figure 2: Schematics of construction of different pseudotyped viruses. 293T cells were transfected with pREC_cplt_R/U5/gag/Δpol, pREC_HIV_env
(or pMD.G), and R8.91 at a mass ratio of 3:1:3 to produce: pseudoviruses containing HIV-1 cplt_R/U5/gag/Δpol sgRNA wrapped with HIV-1 envelope
(Virus #1) or VSV-G envelope (Virus #2), with pREC_nfl_NL4-3 alone to produce pseudovirus containing HIV-1 nfl sgRNA with HIV-1 envelope (Virus
#3), with pMD.G and pREC_nfl_NL4-3 to produce pseudoviruses containing HIV-1 nfl sgRNA with both HIV-1 and VSV-G envelopes (Virus #4), and
with pREC_cplt_R/U5/gag/Δpol and pREC_nfl_NL4-3 to produce pseudoviruses containing both HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs wrapped
with HIV-1 envelope (Virus #5).
with virus #1and #3, or #2 and #4 (MOI 10) were also performed
(Figure 4A).
To assure that the two different viral cores entered the same
cell, so that the cplt_R/U5/gag/Δpol and nfl_NL4-3 sgRNAs could
stay together to complement one another during the reverse
transcription process, we fused the two virus particles with
liposome (Effectene, Qiagen). Virus #1 and #3, as well as Virus
#2 and #4, were mixed together with equal amount of viruses,
and then incubated with 7μl of Effectene per ml of virus at room
temperature for 6 hours. The fused virus particles with HIV-1 env
J Aids Imm Res
or VSV-G env were then used to infect U87.CD.CXCR4 cells, and
these were monitored for virus production at different time points
(i.e. Day 3, 5, 10, 14, 17, 24 post-infection) (Figure 7).
Real-time PCR analysis of viral RNA in transfected/infected
cells
Celluar RNA was extracted from U87.CD4.CXCR4 cells infected
with different virus particles and/or transfected with various
vectors using the RNeasy Mini Kit and QIAshredder (Qiagen). cDNA
was produced in the pol region of nfl_HIV-1 and from the tag region
Vol. 1. Issue. 1. 3000101
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
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flanking the 5′LTR in the pREC_cplt_R/U5/gag/Δpol vector using
the following protocol: 5 μL of extracted RNA was added to 2 μL of
antisense primer (20 pmol/μL) and cycled for 88°C for 2 min, 70°C
for 10 min, 55°C for 10 min, 37°C for 10 min, and 4°C hold. Next,
5× first strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), and 10
mM dNTPs were added to each reaction and cycled at 25°C for 10
min, 42°C for 2 min, and a 4°C hold. Finally, MMLV RT (Invitrogen)
was added to the reaction and cycled at 42°C for 1 h, 70°C for 15
min, and a 4°C hold. Beta-globin was also reverse-transcribed from
cellular RNA using random primers as a control [18].
Taqman real-time PCR was performed on the cDNA that was
described in the paragraph above. Briefly, 5 μL of a 1:5 dilution of
cDNA was added to 1× Taqman Universal PCR Master Mix (Applied
Biosystems, Foster City, CA, USA) and the appropriate primers
(300 nM/reaction) and probes (100 nM/reaction) to specif­ically
detect either the pol or tag region. Probes were labeled with FAM/
MGBNFQ (Applied Biosystems) and were designed in part using
Primer Express software (Applied Biosystems) [18]. Samples were
run on an ABI PRISM 7700 sequence detection system in a 96-well
format (50°C 2 min, 95°C 10 min, and 40 cycles of 95°C for 15 s.
and 58°C for 1 min) and analyzed using SDS 1.9.1 software (Applied
Biosystems). Samples were quantitated based on cDNA standards
run alongside the samples with known copy numbers based on
RNA concentration. To create cDNA standards, a region from pNL4–
3 pol and a region of pREC_ cplt_R/U5/gag/Δpol were cloned into
the pCR2.1-TOPO vector (Invitrogen) and transcribed with T7
polymerase using a MEGAscript transcription kit (Ambion, Austin,
TX, USA). RNA produced during transcription was quantitated
and diluted by 10-fold serial dilutions to create standards prior
to reverse transcription. RNA was reverse-transcribed using the
same protocol to make cDNA which was then used as standards in
real-time PCR alongside the samples. β-globin was also amplified
by real-time PCR from cDNA of cell lysates as a way to standardize
cDNA input into the real-time PCR reactions.
defects of HIV-1 caused by mutations in the capsid can be efficiently
restored when pseudotyped with the VSV-G envelope glycoprotein
[25], suggesting that the VSV-G pathway might provide some kind of
protection of the viral genome from degradation by host enzymes
before/during reverse transcription. Based on these studies, we
generated two additional viruses pseudotyped with VSV-G. Virus #4
was the same as virus #3 but additionally pseudotyped with VSV-G.
Finally, virus #5 served as a positive control and harbored both the
HIV-1 nfl and cplt_R/U5/gag/Δpol sgRNAs. Since the viral RNAs
differ in sequence, we could perform RT-real-time PCR to detect
the sgRNA copy numbers in viruses by using probes specific for the
two sgRNAs [18]. As showed in Figure 3A, viruss #1 and #2 only
harbored the cplt_R/U5/gag/Δpol sgRNA, viruses #3 and #4 had
only nfl sgRNA, virus #5 was positive for both sgRNAs, and the two
sgRNAs were packaged into virus particles at similar efficiencies
(i.e. 109-10 copies per ml culture).
A virtual TCID50 assay was performed on all virus particles
using a methodology previously described [21]. Briefly, the serial
dilutions of virus stocks followed by RT activity assay had a highly
significant direct correlation of viral RT value to the infectious
titer (TCID50 value) of same virus stocks. RT activity can served
as a surrogate for viral RNA copies or p24 antigen content for over
50 primary HIV-1 isolates. Since virus #5 is infectious but harbors
the same virus components of virus #1-4, we compare the serial
dilutions of RT activity of the latter with the infections titer of virus
#5 (Figure 3B), which was used to normalize the amount of virus
input for the infection.
There is no virus production following dual infection with the
two complementary virus particles
In a previous study [18], we demonstrated that virus #5
Results
Construction of virus particles harboring partial HIV-1 genomes
In order to investigate the role of HIV-1 viral core in the process
of reverse transcription, we devised a methodology to produce
several virus particles harboring different HIV-1 subgenomes
that require trans-complementation between these viral sgRNAs
to complete reverse transcription and generate productive virus
infections. As described in Figure 2, virus #1 was produced from
293T cell co-transfected with the pREC_cplt_R/U5/gag/Δpol to
produce and package the cplt_R/U5/gag/Δpol complementing
sgRNA, the pΔR8.91_gag/pol to produce the core proteins and viral
enzymes, and pREC_HIV_env to pseudotype these virus particles
with HIV-1 Envelope glycoproteins. pREC_cplt_R/U5/gag/Δpol is a
new version of cplt vector containing HIV-1 R, U5, gag and partial
pol sequences, which has higher packaging efficiency [22]. Virus #2
was the same as virus #1 in that it only encapsidated the cplt_R/
U5/gag/Δpol RNA but was psuedotyped with VSV-G (from pMD.G)
rather than with HIV-1 Env. Virus #3 was produced from 293T
cells transfected with pREC_nfl_ NL4-3 to generate a nfl sgRNA for
encapsidation as well as produce the entire HIV-1 proteome for
proper virus particle assembly.
The endocytic pathway through clathrin-mediated endocytosis
is a basic mechanism by which the virus core is delivered into the
target cell; this pathway is employed by many kinds of viruses
including influenza viruses, adenoviruses, parvoviruses, and the
Sindbis virus, etc. [23,24]. Incorporation of VSV-G allows viruses
to enter the target cell using the endocytic low pH pathway. RT
J Aids Imm Res
Figure 3: Production and titration of five pseudotyped viruses. (A)
Real-time RT-PCR detection of cplt_R/U5/gag/Δpol and nlf sgRNAs in
virus particle #1, #2, #3, #4, and #5 produced from transfected 293T
cells. (B) RT assay detection of reverse transcriptase activity in the
five different virus particles.
Vol. 1. Issue. 1. 3000101
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
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(containing both HIV-1 cplt and nfl sgRNAs) could successfully
complete reverse transcription and produce a wild type proviral
DNA for integration and subsequent virus production. In this
study, we utilized the same nfl sgRNA and an improved version of
cplt sgRNA (cplt_R/U5/gag/Δpol) with higher packaging efficiency
to investigate whether the two complementary HIV-1 genomes
packaged into separate virus particles could still complement
each other for reverse transcription following dual infection of
susceptible U87.CD4.CXCR4 cells. The two virus particles (virus
#1 and #3 [Condition I], or virus #2 and #4 [Condition II]) were
normalized based on their virtual TCID50, and co-infected U87.
CD4.CXCR4 cell culture with 10 MOI (Figure 4A) (Please note
that the higher MOI caused significant rapid cell death). The postinfection cultures were monitored over 14 days by RT activity in
the supernatant. We performed RT-real-time PCR and quantify
both the cplt_R/U5/gag/Δpol and nfl sgRNAs in the cytoplasm
of U87.CD4.CXCR4 cells at 48 hours post-infection/transfection.
The results showed both of sgRNAs successfully entered into the
susceptible cells with similar amount of the two sgRNA species
in condition I, II, and the positive control (i.e. monoinfection with
virus #5, containing both nfl and cplt_R/U5/gag/Δpol sgRNAs
within one viral core) (Figure 4B), however, neither the HIV
envelope glycoprotein nor VSV-G pseudotyped-virus co-infection
of U87.CD4.CXCR4 cells established viral infection in two weeks’
period of culture, while the infection with virus #5 produced
infectious viruses shortly after the inoculation (Figure 4C). We
have previously shown that the cplt RNA, a previous version of
cplt_R/U5/gag/Δpol can initiate reverse transcription and act as
a template for (-) strand strong stop DNA synthesis [18]. Here, we
again verified that reverse transcription could be initiated in both
virus #1 and virus #2 by detecting (-) strong stop DNA product at
12 h post infection (data not shown).
Free sgRNA in the cytoplasm could not help the complementary sgRNA in the HIV-1 core to complete reverse transcription via either HIV-1 envelope glycoproteins or endocytic,
VSV-G mediated pathway
Considering that the above non-infection of the culture was
possibly due to the low chance of co-infection of two different
viruses with 10 MOI in the same cells (please note the lower MOI
did not produce infectious virus, either. Data not shown), the
plasmids were transfected into U87.CD4.CXCR4 cells to generate
either cplt_R/U5/gag/Δpol or nfl sgRNA in the cytoplasm, followed
by the infection of the same cells with the viruses containing the
corresponding complementary sgRNA. This strategy ensures plenty
of at least one of the sgRNAs in the cytoplasm that will significantly
increase chance to meet another sgRNA in the partially dismantled
viral core or in the cytoplasm.
Figure 4: Detection of HIV-1 production in dual infection where the
complementary viral subgenomes were located in different capsids.
(A) Three different coexisting patterns of the complementary HIV-1
cplt_R/U5/gag/Δpol and nfl sgRNAs: sgRNAs are in different viral cores
wrapped with HIV envelopes (Condtion I); sgRNAs are in different viral
cores wrapped with VSV-G envelopes (Condition II); and sgRNAs are in
same viral core wrapped with HIV envelopes as a positive control. (B)
Real-time RT-PCR detection of sgRNAs in U87.CD4.CXCR4 cells infected
by viruses in different combinations. (C) RT assay detection of HIV-1
production post-infection of U87.CD4.CXCR4 cells.
J Aids Imm Res
Through different DNA transfection and virus exposures of
U87.CD4.CXCR4 cells, we provided the HIV-1 nfl and cplt sgRNAs
in two contexts: (A) cplt_R/U5/gag/Δpol sgRNA produced from
transfected pREC_cplt_R/U5/gag/Δpol plasmid and nfl RNA
derived from infection with virus #3 (Condition III), and (B) nfl RNA
from transfected pREC_nfl_ NL4-3 plasmid and cplt_R/U5/gag/
Δpol RNA from virus #1 (Condition IV) (Figure 5A). By using realtime PCR, in condition III, we detected 104-5/ml of nfl sgRNA and
significant higher level of cplt_R/U5/gag/Δpol sgRNA (~109/ml)
in the cell culture. Similarly, in condition IV, significant higher level
of nfl sgRNA were also detected. The post-infection/transfection
cultures were again monitored over 14 days by RT activity in the
supernatant. However, there was no infectious virus produced
from either cultures (Figure 5C). These findings suggest that,
in HIV envelope entry pathway, the free subgenomic RNA, either
cplt_R/U5/gag/Δpol or nfl, in the cytoplasm could not help the
complementary sgRNA in the HIV-1 core to complete the reverse
transcription process.
We then used VSV-G pseudotyped viruses (i.e. virus #4,
containing nfl sgRNA, or #2, containing cplt_R/U5/gag/Δpol
sgRNA) to infect the U87.CD4.CXCR4 cells which expressed
cplt_R/U5/gag/Δpol or nfl sgRNA through transfection of the
corresponding plasmids (Condition V or VI). RT-real time PCR
results again indicated that the two complementary viral genomes,
Vol. 1. Issue. 1. 3000101
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
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Complementation of reverse transcription via fusion of
VSV-G pseudotyped virus particle prior to virus entry
In a previous study, we discovered that co-packaging of the
cplt and nfl RNA subgenomes into the same virus particle resulted
in complementation of the reverse transcription process and
Figure 5: Detection of HIV-1 production in HIV env pathway where
the complementary viral subgenomes were separated in cytoplasm
and capsid. (A) Three different coexisting patterns of HIV-1 cplt_R/
U5/gag/Δpol and nfl sgRNAs: cplt_R/U5/gag/Δpol sgRNA produced
in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_
cplt_R/U5/gag/Δpol vector, and nfl sgRNA located in viral core through
HIV-1 envelope-mediated entry (Condition III); nfl sgRNA produced in
cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_nfl_
NL4-3 vector (Condition IV), and cplt_R/U5/gag/Δpol sgRNA located
in viral core through HIV-1 envelope-mediated entry; and both sgRNAs
are in same viral core wrapped with HIV envelopes. (B) Real-time RTPCR detection of sgRNAs in U87.CD4.CXCR4 cells infected/transfected
in different combinations. (C) RT assay detection of HIV-1 virus
production post-infection/transfection of U87.CD4.CXCR4 cells.
cplt_R/U5/gag/Δpol and nfl sgRNAs, were found in U87.CD4.CXCR4
cells (Figure 6B). However, this strategy did not produce infectious
virus, either (Figure 6C). This result suggested that the endocytic
pathway for entry again did not appear to permit the free sgRNA
in the cytoplasm to the complementary sgRNA in the viral core to
complete the reverse transcription process.
J Aids Imm Res
Figure 6: Detection of HIV-1 production in VSV-G env pathway where
the complementary viral subgenomes were separated in cytoplasm
and capsid. (A) Three different coexisting patterns of HIV-1 cplt_R/
U5/gag/Δpol and nfl sgRNAs: cplt_R/U5/gag/Δpol sgRNA produced
in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_
cplt_R/U5/gag/Δpol vector, and nfl sgRNA located in viral core
through VSV-G/HIV-1 envelope-mediated entry (Condition V); nfl
sgRNA produced in cytoplasm of U87.CD4.CXCR4 cells through
transfection of pREC_nfl_NL4-3 vector, and cplt_R/U5/gag/Δpol
sgRNA located in viral core through VSV-G envelope-mediated entry
(Condition VI); and both sgRNAs are in same viral core wrapped with
HIV envelopes as a positive control. (B) Real-time RT-PCR detection
of sgRNAs in U87.CD4.CXCR4 cells infected/transfected in different
combinations. (C) RT assay detection of HIV-1 virus production postinfection/transfection of U87.CD4.CXCR4 cells.
Vol. 1. Issue. 1. 3000101
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
Page 7 of 9
production of fully infectious, wild type virus and thus showed that
they could help one another produce infectious viruses. However,
in the present study, the two complementary viral genomes, nfl
and cplt_R/U5/gag/Δpol RNA, were wrapped into separate capsids
within separate virus particles. As described above, the failure of
establishment of infection through dual infection is possibly due to
the low chance for the two different viral cores to meet each other
in the cytoplasm. However, the transfection/infection strategy
should have provided plenty of chance for the two sgRNA to meet
each other if the transfection-produced sgRNA could penetrate the
partially dismantled viral core, or the sgRNA in the virus particle
released from the viral core (note the cplt sgRNA containing virus
could initiate the reverse transcription, resulting in the breakdown
of its viral core) which should result in productive infection if the
reverse transcription could occur in the cytoplasm. Therefore,
it suggested that the viral core is indeed important for the HIV-1
reverse transcription process and the reverse transcription could
not occur in the cytoplasm.
To further investigate the possibility of viral core fusion during
the reverse transcription process, we mixed the two viral cores
through inoculating virus #1 with #3 (condition VII, HIV envelope
entry pathway) or virus #2 with #4 (condition VIII, VSV-G envelope
entry pathway) in the presence of liposome overnight and then
infected U87.CD4.CXCR4 cells (Figure 7A). This procedure likely
generates large enveloped virus-like particles with a membrane
surrounding two or more viral cores. If these cores mix and
form a new functional viral core, complementation of reverse
transcription could occur with de novo infection and result in
production of replication competent virus. Surprisingly, when
the VSV-G pseudotyped particles (condition VIII) were fused with
liposome, RT activity was detected in the supernatant by day 10
and peaked at day 14 post-infection (Figure 7B). This virus was
successfully subsequently passaged on new U87.CD4.CXCR4 cells.
However, the fused HIV enveloped virus (condition VII) didn’t
show productive virus infection. The core mixing should have
occurred in both conditions VII and VIII considering that liposome
treatment is unlikely to favor more fusion in one condition over
the other. Within the fused virus particles, it is unlikely that the
virus cores from either condition would have different properties.
Because the reverse transcription could not occur in the cytoplasm
after viral core breakdown, these results suggest that, in the HIV
envelope pathway, before and after the multiple core containing
virus entry into the host cell, the viral cores did not fuse to form
new functional viral cores, instead, they broke down, resulting in
the degradation of sgRNAs by hostile cellular enzymes in the host
cytoplasm. In contrast, in the VSV-G pathway, the virus particles
were endocytosed through VSV-G envelope glycoproteins to form
early endosomes where the sgRNAs were released from the viral
cores and accomplished the reverse transcription process in an
unknown mechanism (Figure 7A).
Discussion
The events that take place after entry of the HIV-1 viral core
are somewhat unclear. It was once held that the core uncoated
immediately post-fusion and release into the cytoplasm [1214]. However, some studies showed that TRIM5α can bind to
polymeric CA, resulting in premature core disassembly and
mediation of the RTC to proteosomal degradation, thus blocking
reverse transcription [26]. It has also been shown that mutations
that reduce the stability of the viral core are deleterious for viral
infectivity and reverse transcription [27-31]. Other studies showed
that the incoming capsid seems to retain an intact structure during
its journey from the cell surface to the nucleus, reverse transcription
happened within an integral capsid shell, and uncoating occurs at
J Aids Imm Res
Figure 7: HIV-1 virus production in dual infection with fused virions.
(A) Virus particles containing the complementary HIV-1 viral sgRNAs
(i.e. virus #1 and #3, virus #2 and #4) were fused through liposome,
and then infected the target cells via either HIV-1 or VSV-G envelope
pathway. (B) RT assay detection of HIV-1 virus production in U87.
CD4.CXCR4 cells infected with fused viruses through HIV-1 or VSV-G
envelope pathway.
the nuclear pore upon completion of RT [16,25,15,32]. Thus, viral
DNA synthesis and routing of HIV-1 RTCs/PICs to the nuclear
membrane might be two independent events. Uncoating of the HIV1 core is possibly the last step before the PICs across the nuclear
pore, taking place after the DNA Flap formation that promotes it [8,
12,32,33]. This implies that the HIV-1 viral capsid might also play
an important role in transporting the PICs into the nucleus.
In the present study, we modified our previously constructed
unique system [18] to specifically explore the function of the viral
capsid during HIV-1 reverse transcription, whether the reverse
transcription can occur in the host cytoplasm, and whether the
different entry pathway will cause different consequence postviral entry. The HIV-1 NL4-3 genome was split into two parts, nfl
and cplt_R/U5/gag/Δpol sgRNAs, that are complementary to each
other and can accomplish reverse transcription via strand transfer.
Various virus particles with functional viral capsids were created
to contain the two sgRNAs and wrapped with HIV-1 or VSV-G
envelopes. We then set up several different coexisting patterns for
the two sgRNAs in the susceptible cells through dual infection in
the presence or absence of liposome, or infection/transfection (i.e.
one sgRNA in the viral core, and the other sgRNA in the cytoplasm,
or both in the viral core together or separately). The advantage of
this system is that the two complementary HIV-1 sgRNAs can be
separated in their specific locations; we can thereby distinguish
Vol. 1. Issue. 1. 3000101
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
Page 8 of 9
whether reverse transcription occurs in the viral core, in the
cytoplasm, or in the fused viral core.
If reverse transcription occurs in the cytoplasm, the RNA genome
(either cplt_R/U5/gag/Δpol or nfl) within the viral core should be
released, meet its complementary genome in the cytoplasm. The
two genomes should then cooperate to complete the process of
reverse transcription. However, none of our coexisting patterns with
the HIV-1 sgRNAs located in the cytoplasm (through transfection)
showed production of infectious virus, suggesting that there was no
successful completion of reverse transcription in the cytoplasm.
On the other hand, the viral cores might fuse, resulting in
meeting of the two sgRNAs and completion of reverse transcription
within the fused viral core. However, our results showed that the
co-infection with two sgRNA-containing viruses didn’t produce
any infectious virus, even the fused virions resulting in two sgRNAcontaining viral cores confined in one virus particle did not produce
infectious virus, either. Thus, the viral cores do not appear to be
capable of fusing to complete the first-strand transfer during
reverse transcription. Among all of the coexisting patterns, only
two sgRNAs located in the same capsid can produce replication
competent viruses through the HIV-1 envelope pathway. This serves
as confirmation that structural integrity of viral core is necessary for
successful viral reverse transcription in HIV-1 replication. The study
on the function of the HIV core suggested that the isolated core is
active for reverse transcription [34]. In this study, since the cplt_R/
U5/gag/Δpol RNA-containing virions (virus #1 and #2) contain
sgRNAs from R to the middle of the pol region, including the primer
binding site (PBS), and all of the viral proteins, reverse transcription
was initiated but not completed. It will be interesting to investigate
whether these viral cores are uncoated at this point in the process.
Pathways that the HIV-1 viral capsid may follow post-fusion
or endocytosis into the host cell are not clearly defined. There is
evidence that the cellular cytoskeleton facilitates early steps of
HIV-1 infection [4], and that the capsid is one of the main targets of
regulation. The viral cores are deposited in the cell cytoplasm after
entry via fusion through the HIV-1 envelope. The capsid cores of
HIV-1 and similar retroviruses bind dynein motors and traffic along
microtubules towards the nucleus. On the contrary, HIV-1 virions
carrying VSV-G are predicted to enter cells through an endocytic
pathway, since this is the normal route of VSV infection. These
chimeric viruses are capable of infecting a wide variety of cell types
and were recently used to enhance the utility of HIV-1-based gene
delivery vectors [35]. By tracking fluorescent viral complexes in
living cells infected with the VSV-G pseudotyped virus, researchers
demonstrated that short distance and rapid movements of HIV-1
cores are characteristic for an actin-polymerization-dependent
transport [19,32]. The microtubule network also supports long
distance movement of HIV-1 core complexes. However, actin-cabledependent trafficking systems recruited by HIV-1 complexes that
are delivered through HIV Env and VSV-G may differ. Indeed, HIV-1
infection is blocked at the RT level in cells that express siRNAs to
the Arp2/3 actin nucleator complex, which inhibits polymerization
of actin. This block is no longer observed when using VSV-G
pseudotyped HIV-1 [36].
Viruses entering target cells through the VSV-G pathway will
first form endosomes and then lysosomes (which contain enzymes),
and virions will be limited within a closed subcellular structure.
Brun S. et al.[25] found that HIV-1 viruses bearing mutated capsids
that disrupt virus infectivity via impairment of core assembly
and stability could be efficiently restored by pseudotyping with
the VSV-G envelope glycoprotein through the endocytic pathway
instead of the HIV-1 Env-mediated fusion process [25]. This
indicates that the pathway that viral core shunting in the target cell
J Aids Imm Res
may influence subsequent function of the viral core, and possibly
protect HIV-1 RNA from degradation by host enzymes. Our present
study found that, even though co-infections with the two different
VSV-G-viruses didn’t produce any infectious virus, infectious
progeny was successfully created when the two virions were fused
by liposomes before infection, which ensured the two cores entered
the host cell together. This result indicates that defects in reverse
transcription can be restored using the VSV-G mediated endocytic
pathway, but the two capsids must be in close proximity in order
for this to happen. Because it has been demonstrated that the
reverse transcription cannot occur in the cytoplasm and the viral
cores cannot fuse to form new functional viral cores, it is highly
likely that either the two viral cores/capsids are broken down in
the closed subcellular structure (i.e. endosome/lysosome), or that
the uncoating is triggered by reverse transcription, resulting in the
release of the two complementary HIV-1 RNA subgenomes and
completion of the first-strand transfer. On the other hand, the fused
virus bearing HIV-1 envelopes may be set apart post-entry and
degraded by the host enzymes in the cytoplasm, resulting in nonproductive infection. Therefore, an intact state of the viral core is
important for viral reverse transcription in natural HIV-1 infection,
and uncoating might only entail the opening of viral core, rather
than complete degradation of the capsid, so that the necessary
materials can access the reverse transcription machinery.
Overall, HIV-1 reverse transcription is a complex and highly
ordered process. HIV-1 RNA is vulnerable and easily broken when
exposed to the numerous enzymes in the cytoplasm; its viral core is
thus an ideal site to protect the viral RNA and confine the necessary
materials within a limited space in order to ensure successful
reverse transcription. The number of reverse transcriptase per
capsid (80 to 120) in the HIV-1 reverse transcription complex
(RTC) makes this especially true. Considering the need to maintain
stoichiometry in the reactions between enzymes and viral templates
for rate limiting steps such as polymerization pauses, strand
transfers, and formation of the central DNA Flap, it is only logical
that reverse transcription occurs within an integral viral core/
capsid structure in the cytoplasm of host cells [37-39]. Even if the
VSV-G pathway helps the capsids that contain two complementary
genomes complete reverse transcription via strand transfer, a
relatively enclosed space (i.e. lysosome) is necessary for the firststrand transfer. The role of CA in PIC production and transport
towards the nucleus still needs elucidation.
Acknowledgements
This study was supported by research grants awarded to
Y.G. and E.J.A. (NIH/NIAID AI84816 and AI49170), and to W.H
(Y201023, Department of Public Health of Jiangsu Province, China).
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*Corresponding author: Yong Gao, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave,
Cleveland, Ohio 44106, USA, E-mail: [email protected]
Received Date: August 08, 2014, Accepted Date: February 24, 2015, Published Date: March 02, 2015.
Copyright: © 2015 Weining Han, et al. This is an open access article distributed under the Creative Commons Attribution License, which ermits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human
Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.
J Aids Imm Res
Vol. 1. Issue. 1. 3000101