Journal of General Virology (2015), 96, 3382–3388
Short
Communication
DOI 10.1099/jgv.0.000265
Properties of human immunodeficiency virus
type 1 reverse transcriptase recombination
upon infection
Sayuri Sakuragi, Tatsuo Shioda and Jun-ichi Sakuragi
Correspondence
Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, Japan
Jun-ichi Sakuragi
[email protected]
Received 9 July 2015
Accepted 13 August 2015
Reverse transcription (RT) is one of the hallmark features of retroviruses. During RT, virusencoded reverse transcriptase (RTase) must transfer from one end to the other end of the viral
genome on two separate occasions to complete RT and move on to the production of proviral
DNA. In addition, multiple strand-transfer events between homologous regions of the dimerized
viral genome by RTase are also observed, and such recombination events serve as one of the
driving forces behind human immunodeficiency virus (HIV) genome sequence diversity. Although
retroviral recombination is widely considered to be important, several features of its mechanism
are still unclear. We constructed an HIV-1 vector system to examine the target sequences
required for virus recombination, and elucidated other necessary prerequisites to harbour
recombination, such as the length, homology and the stability of neighbouring structures around
the target sequences.
Retroviruses possess a ssRNA genome and a reverse transcriptase (RTase) within its virion. Strand-transfer during
reverse transcription (RT) is one of the characteristic features of retroviruses. After retroviral infection into the
host cell, the RTase starts synthesizing negative-stranded
DNA from the primer binding site, where the tRNA
primer anneals. After two strand-transfer events at the
end of the viral RNA genome, positive-strand DNA
synthesis commences, and the proviral dsDNA, which has
two LTRs at the each end of the strand, is produced
from the viral RNA template.
As the viral genomes in the retroviral virion always form
dimers, genomic recombination events between the two
strands are frequently observed during the RT process.
This recombination of the HIV genome is believed to
have some merits for virus survival, as it is a mechanism
to impart genetic variation to its offspring (Malim & Emerman, 2001). The recombination occurs by the transfer of
RT between homologous regions of two separate RNA
strands during the first positive-strand DNA synthesis.
It has been reported that this strand-transfer occurs
approximately once every 1–2 kb and there are reported
‘hot-spot’ regions that tend to be common targets for
this strand transfer to occur (An & Telesnitsky, 2002b;
Galetto et al., 2006; Moumen et al., 2003).
The occurrence of HIV genome recombination appears to
be an inevitable event, and reports are being published
daily describing circulating recombinant forms of the
Supplementary methods are available with the online Supplementary
Material.
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virus; however, the mechanisms that underlie this recombination event are still not completely understood (Coffin,
1979; Delviks-Frankenberry et al., 2011; Galetto & Negroni,
2005; Moore et al., 2009). In this report, we utilized an
HIV-1 vector system that reconstituted a fluorescent protein upon genomic recombination and we looked for
characteristics around the target sequence that are involved
in this genomic recombination event. We examined the
minimal target length required for RT strand transfer and
the extent of required target sequence homology, and studied how surrounding genomic sequences may be involved
in the efficiency of recombination.
RT-mediated retroviral genomic recombination is
suggested to occur by utilizing mechanisms similar to
those of homologous recombination, although the two
mechanisms are not identical (Hu & Temin, 1990).
During minus-strand DNA synthesis, a DNA–RNA
hybrid that contains the template viral genome RNA and
the newly synthesized DNA is produced. Viral RT-encoded
RNaseH digests the RNA portion of this hybrid and creates
a ssDNA overhang, which is complementary to the viral
RNA template (the donor region). The ssDNA overhang
can anneal to the homologous sequence of a second viral
RNA strand (the acceptor region). After the overhand
and its homologous sequence anneals, the RT has an
opportunity to transfer its template to the second strand
of RNA, which is located near to the first RNA strand, as
they are in a dimerized state (review by Galetto & Negroni,
2005; Hu & Hughes, 2012; Hu & Temin, 1990).
An assay system to measure genome recombination rates
during HIV-1 RT upon infection was constructed as
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HIV-1 reverse transcriptase recombination on infection
(a)
NLrNnG* system (* = duplicated length: 30, 40, 50, 60, 120, 250 bases)
rev
gag
rev
vif
nef
Nhel
tat
LTR
tat
vpr
pol
LTR
env
vpu
G
HSA
F
F
P
Spacer S1
FL2-H
Vector with
no marker
100 101 102 103 104
FL1-H
GFP
NLrHnG*S1
100 101 102 103 104
100 101 102 103 104
HSA
FL2-H
(b)
A
B
100 101 102 103 104
FL1-H
GFP
B/(A+B)×1000/* = recombination times kb–1
(c)
G
F
F
P
1.8
1.6
Recombination Times/kb
1.4
1.2
1
0.8
0.6
0.4
0.2
0
G30S1
G40S1
G50S1
G60S1
G120S1
G250S1
Fig. 1. Recombination measurement system for HIV-1. The replication-competent HIV-1 proviral clone pNL4-3 (Adachi et al.,
1986) was used as a progenitor for the proviral mutant constructs described below. The vector–GFFP contained the overlapping GF and FP fragments of the genes for GFP separated by spacers (for details, see Supplementary methods, available in
the online Supplementary Material). (a) Schematic figure of the GFFP vector (NLrHnG*). The vpr gene was replaced by murine HSA. Various GFFPs were inserted at the position of the nef gene. (b) Representative data observed by FACSCalibur.
The HIV-1 recombination assay was performed based on the direct repeat deletion assay developed by Svarovskaia et al.
(2000) and our previous work (Sakuragi et al., 2008) with some modifications (see Supplementary methods). The cells were
analysed on a FACSCalibur (BD Biosciences). The production of HSA and GFP were detected and the indicated formula
was applied for calculation of recombination rate [recombination times (kb RNA)21]. In short, HSA production represents the
infection efficiency of the vector and GFP production represents the occurrence of recombination during one cycle of
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S. Sakuragi, T. Shioda and J.-I. Sakuragi
infection. As recombination to restore the GFP gene only occurs at the duplicated region (‘FF’ in GFFP), the recombination
rate could be calculated by dividing the recombination efficiency (GFP/HSA) by the nucleotide length of the duplicated region
(kb). (c) Required RNA length for recombination. The 39 ends of the duplicated regions (30–250 bp) of eGFP were inserted
by Acc I restriction enzyme site (452 nt from the ATG of eGFP). The numbers in the samples on the x-axis (‘*’ of G*S1) represent the nucleotide lengths (bp) of the duplicated region. At least three independent experiments were performed and the
mean¡SEM is presented.
described below, with reference to previous reports (Nikolenko et al., 2004, 2005). The murine heat-stable antigen
(HSA) gene and an engineered eGFP gene (GFFP), which
served as biomarkers, were inserted into the positions of
the viral vpr and nef genes, respectively (Fig. 1a). The
coding frame of eGFP was separated by spacer nucleotides
(of non-viral origin) and 30–250 bp sequences of the eGFP
gene were engineered to bookend the spacer nucleotides
(S1) as ‘donor’ and ‘acceptor’ regions for the template
transfer event, which led to a variety of reconstituted
GFFP genes. During RT, the repeated ‘F’ portion may be
deleted via recombination to reconstitute a fully functional
GFP. HSA gene expression served as a marker for virus
infection, while eGFP expression served as a marker for
the recombination event that occurs during RT. The
expression of these biomarkers was analysed by FACSCalibor (BD Biosciences) and the recombination rate (kb
RNA)21 was calculated using the equation shown in
Fig. 1(b).
To determine the effect of target length on recombination,
we analysed the length of the homologous sequence
required for HIV-1 recombination. A series of GFFP
genes that contained various lengths of duplicated
sequences (30, 40, 50, 60, 120 or 250 nt) with the S1
spacer was constructed and used in the recombination
assay (Fig. 1c). Very little recombination was observed in
the 30 nt (G30) mutant, and the frequency of recombination gradually increased as the duplicated sequence length
became longer. Recombination reached maximum efficiency in the G60 mutant, and successively longer homologous sequences did not further affect the efficiency. These
results suggested that a minimum homologous sequence
length of 60 nt is sufficient to support HIV-1
recombination.
We next tried to examine how the extent of homology at
the target site might affect HIV-1 recombination. Based
on the G60 mutant, various silent substitution mutations
were introduced to disrupt recombination. Four nucleotide
substitutions were introduced in three ‘95 %’ mutants,
seven substitutions in one ‘90 %’ mutant, 13 substitutions
in one ‘80 %’ mutant and 19 substitutions in two ‘70 %’
mutants (Fig. 2a). The silent mutations of 95 %2 and
95 %3 mutants were situated in the 59 or 39 half of the
duplicated region, respectively, whereas those of the
95 %1 mutant were evenly spaced throughout. The result
of the assay suggested that 95 % sequence identity with
the WT was sufficient to maintain recombination efficiency, whereas lower sequence identities rapidly diminished the efficiency (Fig. 2b). In the 90 % mutant,
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recombination efficiency dropped to one-third of that of
the WT and infrequent recombination was observed in
the mutants with 80 % or less identity. Interestingly, even
in the 95 % mutants, mutations needed to be spaced
evenly throughout, rather than on both ends of the
sequence, for efficient recombination.
Finally, we modified the sequences of the spacer regions
between duplicated sequences in order to observe their
role in recombination. We originally employed the multicloning site of plasmid pGEM5Zf(+) (Promega) as the
spacer. Its length is about 250 bp and it contains many
palindromic sequences (restriction enzyme recognition
sites). Therefore, it forms relatively stable secondary structures with a DG of nearly 280 (Fig. 3a). We further constructed two series of GFFP mutants, one containing no
spacer (NS) and where the duplicated regions of GFFP
were set directly side by side and the second series utilizing
anti-CD52 as the spacer (S3). S3 is about 250 bp, which is
equivalent to the coding region of the murine CD52
gene, but arranged in reverse orientation. The secondary
structure of this spacer region is relatively unstable
(DG541.88) (Fig. 3a). We constructed the 60 and 120 bp
GFFP mutants containing NS and S3 and compared the
effect on recombination efficiency (Fig. 3b). The recombination rate was dramatically reduced in both the NS
and S3 constructs, thus suggesting the importance of the
sequence upstream of the strand transfer point.
In this report, we employed a system to measure the
strand-transfer-mediated recombination efficiency upon a
single retroviral vector infection (Fig. 1). Using this
system, we focused on the relationship between the
‘donor’ and ‘acceptor’ RNA regions of the template and
analysed several factors that may be required for recombination. The minimally required length of homologous
sequences for strand transfer appeared to be 60 bases
(Fig. 1c). The strand transfer must be very strictly controlled, as it must occur at the R region of the LTR at
both ends of the genome during the first and second
strand transfer. Furthermore, it must occur between the
same positions of the two genomic copies and must not
occur between positions outside of the R region to prevent
deletion or duplication of the gene during RT. The length
of these R regions is about 100 bases, while the next longest
homologous regions within the genome are the 20 base
polypurine tracts, which are located at the middle and
39 end of the genome. Thus, 60 bases is a reasonable minimal length for the homology region, as 100 bases are sufficient as a target for strand-switching, whereas 20 bases is
too short to be a target.
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HIV-1 reverse transcriptase recombination on infection
G60
(a)
G
(b)
F
F
P
1.8
1.6
1.4
Times/kb
1.2
1
0.8
0.6
0.4
0.2
0
G60S1
G60/95%1 G60/95%2 G60/95%3 G60/90% G60/80% G60/70% G60/70%2
(c)
G
F
F
P
Strand Transfer
95%1
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95%2
95%3
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S. Sakuragi, T. Shioda and J.-I. Sakuragi
Fig. 2. Required nucleotide sequence homology for recombination. (a) Mutations introduced in the series of mutants. (b)
Measurement of the recombination rate. At least three independent experiments were performed and the average was presented. The error bars represent SEM. (c) Hypothetical scheme of strand annealing during template switching. Solid, light
grey and dark grey lines represent synthesized DNA during RT. The arrowheads represent the 39 end of the DNAs. The lightnings are images of blocks by mutations during homologous recombination.
More than 90 % homology was required between the
donor and acceptor of the 60 base target for efficient
recombination (Fig. 2). These results were consistent with
previous work performed with retroviral vectors carrying
drug-resistant genes (An & Telesnitsky, 2002a, b; Pfeiffer
& Telesnitsky, 2001). In addition, the mutations must be
scattered evenly in the region, as biased concentration of
the mutations dramatically reduced the recombination efficiency. In particular, when mutations were biased to the 39
side of the target, recombination was nearly abolished
(mutant 95 %3). As the first-strand transfer between the
DNA and RNA genome is assumed to occur from the
39 side of RNA (Fig. 2c), this may be a reasonable observation. In case of mutant 95 %2, annealing of the viral
DNA to the 39 half of the acceptor was stable provided
that there were no mismatches. After producing the
RNA–DNA hybrid strand as a scaffold for strand transfer,
the mismatch at the 59 end could be overcome, and recombination could be completed, even at reduced efficiencies.
In contrast, in mutant 95 %3, the scaffold would barely
be established because of the mutations. It was surprising
that the efficiency of mutant 95 %3 was lower than that
of the 90 % mutant, as identical mutations were introduced at the 39 end of the target sequence. One possible
explanation is that the shared sequences between the
95 %2 and 90 % (59 half of the acceptor) mutants may
(a)
attract the template switch to some extent through its secondary structure or the nucleotide sequence itself.
An unbiased mismatch with 90 % identity appears to be a
relatively stringent requirement for recombination
between heterodimerized genomes. As the overall nucleotide identity between HIV-1 and HIV-2 or simian immunodeficiency virus is 70 % or less (Desrosiers, 1990;
Tristem et al., 1992), the production of chimeric virus
would be extremely rare, even if co-packaging of two
species of virus genome were to occur, which in itself is
an extraordinary event (Dilley et al., 2011; Moore et al.,
2007; Ni et al., 2011). Even among HIV-1 subtypes,
sequence identities higher than 90 % are not common
(Gao et al., 1998). However, co-packaging of heterosubtype genomes would occur relatively easily because the
SL1 hairpin-loop sequence is the primary driving force
behind heterodimerization (Sakuragi et al., 2010).
In addition, recombination can still occur between
sequences with less than 90 % identity to a certain
extent (Fig. 2b). Thus, the recombination between HIV-1
subtypes may actually be a common event in nature
(Vuilleumier & Bonhoeffer, 2015).
The stability or structure of the spacer region greatly influenced recombination efficiency (Fig. 3). There has been an
idea proposed to explain the driving force behind
(b)
G
F
F
P
1.6
1.4
1.2
S1: ΔG = –79.56
1
0.8
0.6
0.4
0.2
0
S3: dG = –41.88
G60S1
G120S1
G60NS
G120NS
G60S3
G120S3
Fig. 3. Role of the spacer region between donor and acceptor sites of recombination. (a) Stability and secondary structure of
the spacers. (b) Measurement of the recombination rate. At least three independent experiments were performed and the
mean¡SEM is presented.
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Journal of General Virology 96
HIV-1 reverse transcriptase recombination on infection
recombination called the ‘forced copy-choice model’
(Coffin, 1979; Moumen et al., 2003). According to this
model, the template switch should stall during RTasemediated strand extension, as it must search for a target
to anneal to the newly synthesized DNA. The encounter
of RTase with the RNA terminal is a good opportunity for
stalling and thus efficient strand transfer occurs between
the 59 and 39 R regions. Similarly, encapsidated viral RNAs
are suggested to be rarely intact and contain many nicks,
and thus the template switch may also be primed by nicks
(Coffin, 1979). Our data suggest that the stable structure
of the spacer S1 would also work as a steric barrier for RT
and cause RTase stalling. As the recombination rate of the
vectors with the spacer S1 was close to the rate observed in
epidemiological reports (one event per 1–2 kb) (OnafuwaNuga & Telesnitsky, 2009; Onafuwa et al., 2003), the steric
barrier of an RNA structure such as the spacer S1 might be
a common trigger for recombination. To further support
this hypothesis, there were several detailed works about
HIV-1 strand transfer that have described how stable RNA
stem–loop formation, especially at the top of the hairpin
loop, serves as a recombination ‘hot-spot’ (Galetto &
Negroni, 2005; Moumen et al., 2003; Simon-Loriere et al.,
2009). Our data not only underscore the importance of
RNA sequences in the strand-transfer region but also stress
the importance of structures adjacent to the regions for
recombination.
In conclusion, we examined the characteristics of the
acceptor region required for efficient HIV-1 recombination
and determined several basic but important prerequisites
for retroviral recombination.
Acknowledgement
We greatly appreciate Ken-ichi Yoshida for his assistance of the experiments. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of
Health, Labour, and Welfare, Japan.
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