(Received for publication, July 12, 1995; and in revised form, October 25, 1995)
From the
According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.
HIV ()is a retrovirus and the causative agent of
AIDS. Retroviruses convert their RNA genomes into proviral DNA by the
process of reverse transcription (reviewed in Whitcomb and
Hughes(1992)). Reverse transcription starts from a cellular tRNA that
binds near the 5` end of the virus to the PBS. Synthesis proceeds to
the end, forming the minus strong stop DNA. Transfer of this DNA to the
3` end of either co-packaged RNA molecule is necessary to complete the
(-)-strand DNA. This transfer reaction is thought to be
facilitated by complementary regions at the ends of the virus and by
the RNase H activity that degrades the RNA complementary to the newly
synthesized DNA. Second strand synthesis is initiated from a
purine-rich sequence tract (PPT) located just upstream of the U3
region. This oligoribonucleotide is created by degradation of the viral
genome by the RNase H activity of the RT. Synthesis from the PPT,
ending in copying part of the tRNA primer of the(-)-strand DNA,
forms the plus strong stop DNA. This DNA needs to be transferred to the
3` end of the (-)-strand DNA to resume synthesis. Transfer in
this case is thought to be facilitated by the removal of the tRNA
primer which allows the complementary sequences spanning the PBS site,
at the ends of the (+)- and(-)-strands, to circularize and
complete their synthesis (Fig. 1A). After this
circularization step in HIV, polymerization displaces about 637
nucleotides (Ratner et al., 1985) to generate the long
terminal repeats containing the duplications of the U3, R, and U5
regions. This whole process is carried out by the enzyme reverse
transcriptase (RT), which is encoded by the pol gene and is
carried inside the virion. The native enzyme in HIV is a heterodimer
composed of a 66- and a 51-kDa subunit (diMarzo et al., 1986;
Lightfoote et al., 1986).
Figure 1: Strand displacement substrates. A, the postulated substrate for strand displacement synthesis in the reverse transcription model. B shows the substrates used to study strand displacement in this paper. The substrates were prepared as described under Methods. The template and the 5` end-labeled 19-nucleotide primer were the same in both substrate A and B. The regions labeled as PBS, U5, R, and U3 in the template correspond to the sequences found in the HIV genome. PBS, primer binding site for tRNA. U5 and U3, regions unique to the 5` and 3` end of the LTRs, respectively. R, redundancy common to 5` and 3` ends.
Not only is strand displacement necessary for viral replication, it is also likely to play a role in recombination according to the strand displacement-assimilation model (Junghans et al., 1982). This model proposes that recombination can occur during (+)-strand DNA synthesis. The model assumes that during reverse transcription two (-)-strand DNAs are created, and that (+)-strand synthesis can be initiated at multiple points. According to this model, when a downstream DNA is displaced by upcoming synthesis, this fragment can migrate to, or invade, the second copy of(-)-strand DNA and serve as a primer there. If the fragment of DNA being transferred is not 100% homologous to the new template DNA, it may lead to a recombinant virus. Additionally, strand displacement was proposed to be involved in a new model for the second template switch in reverse transcription (Li et al., 1993). In this model, synthesis from the central PPT displaces the plus strong stop DNA. The displaced DNA can base-pair with the 3` end of the(-)-strand DNA; this would eliminate the need for a second jump or transfer event.
Strand displacement by HIV-1 RT has been documented in vitro by Huber et al.(1989) and Hottiger et al.(1994) for segments up to 50 nucleotides in length, but to date there has been no evidence that this reaction can be extensive enough to carry out the lengthy displacement required in vivo. This activity has also been reported for avian myeloblastosis virus and murine leukemia virus RT (Collet et al., 1978; Matson et al., 1980; Whiting and Champoux, 1994). In murine leukemia virus, there is recent evidence that the enzyme can displace 1,334 base pairs of DNA duplex, which is a much greater distance than what is required during reverse transcription in vivo (Whiting and Champoux, 1994). In this paper, we examine the ability of HIV-RT to displace a 634-nucleotide-long segment of DNA containing the natural LTR sequences in vitro.
The postulated substrate for strand displacement during reverse transcription is shown in Fig. 1A. The substrates used in this study are shown in Fig. 1B. We designed the templates so they would contain the sequences involved during this process in vivo.
Figure 2:
Time course of the strand displacement
reaction. The first two lanes, at the left, contain
products of extension of substrates A and B with T4 DNA polymerase. The
T4 DNA polymerase reactions were carried out using the 10
buffer supplied by the manufacturer. The arrow points to the
position at which T4 polymerase stops synthesis when it reaches the
downstream primer in substrate B. The rest of the lanes contain the
products of standard HIV-RT reactions on substrates A and B. The
reactions were stopped at the times indicated in the figure. The
product sizes were determined using a labeled 100-bp ladder (not
shown).
In the absence of a downstream primer, HIV-RT takes less than 7 min to elongate most annealed primers to the end of the template. After 1 min, the maximum length of extension on substrate A is approximately 200 nucleotides, which gives a rate of synthesis of 3.3 nucleotides per s. Virtually the same distribution of products was obtained after 1 min in two additional independent experiments (not shown). On substrate B, the first elongated primers reach the end after 15 min. Even at the 60-min time point, there is little synthesis to the end of the template. At the 7-min time point, the maximum length of extension is approximately 120 nucleotides, which gives an approximate strand displacement rate of 0.28 nucleotides per s. At 15 min, the maximum length of extension is approximately 250 nucleotides. This gives a consistent displacement rate of 0.27 nucleotide per s. Two other independent experiments produced essentially the same distribution of products (not shown). This indicates that strand displacement slows RT-directed synthesis by approximately 12-fold.
We studied the effect of ionic strength on strand displacement synthesis by titrating the amount of KCl in the reactions from 32 mM to 80 mM (not shown). Synthesis on substrate A was not affected significantly by the amount of KCl in the reactions over the range examined, but synthesis on substrate B was more efficient at low ionic strength. With substrate B at KCl concentrations above 50 mM, there were virtually no extension products longer than 100 nucleotides after incubation with HIV-RT for 30 min. This inhibition of strand displacement at high ionic strength is expected since high salt stabilizes duplex DNA.
Figure 3:
Strand displacement synthesis in the
presence of trap. Trapped HIV-RT reactions were performed for 30 min as
described under Methods. The trap used was
poly(rA)-oligo(dT). Lanes 1 and 4 are
nontrapped reactions, lanes 2 and 5 are trap control
reactions, and lanes 3 and 6 are trapped reactions.
The product sizes were determined using a labeled 100-bp ladder (not
shown).
Even when the strand displacement reactions were carried out for over 3 h in the absence of trap, we did not observe a high proportion of the upstream primer reaching the end of the template (not shown). We thought that extension may not have reached the end of the template because most the enzymes inactivated before synthesis could be completed. To test our hypothesis, we added fresh enzyme to the strand displacement reaction every 30 min and showed that with sufficient time, and a constant replenishment of RT, complete extension of initiated primers can be accomplished (not shown).
NC is a viral protein that normally coats part of the viral genome in vivo. NC promotes strand exchange from double-stranded DNA to single-stranded DNA favoring the most stable complex (Tsuchihashi et al., 1994). NC was titrated into the strand displacement reactions at concentrations ranging from 1.2 µM to 75 nM. These concentrations of NC are enough to cover 200% to 12.5% of the DNA in the reactions. At high concentrations, NC was inhibitory for synthesis, and, at low concentrations, no stimulation of strand displacement was observed (not shown). The effect of NC was tested by adding NC before and after preincubation of the substrate with RT. To investigate whether the effect of NC was due to the melting of the primer away from the template, we fractionated an aliquot of the completed reactions on a native polyacrylamide gel. We found approximately the same proportion of unannealed primers in the presence and absence of NC (not shown).
We then tested whether a protein known to stimulate DNA synthesis by melting template secondary structure could enhance the reaction. E. coli SSB has this capacity, presumably because of its ability to bind and stabilize single-stranded DNA (Kornberg and Baker, 1992). Decreasing concentrations of SSB were added to the strand displacement reactions after preincubation of the substrate with HIV-RT (Fig. 4A). E. coli SSB enhanced the extent of strand displacement synthesis, especially at concentrations of 150 nM and lower (lanes 3-5). In order to quantitate this enhancement, we measured the percent of the extended products longer than 400 nucleotides. There was an approximately 3-fold increase in these products in the presence of E. coli SSB. Furthermore, the percent of extended primers in the presence and absence of E. coli SSB remained constant. This indicates that E. coli SSB affected the reaction by increasing the length of the extension products rather than increasing the amount of extended products. This effect of E. coli SSB was unchanged when the protein was preincubated with the substrate before the addition of the RT. When E. coli SSB was present at 600 and 300 nM (lanes 1 and 2), the enhancement of strand displacement synthesis was diminished. This could be because E. coli SSB aggregates at high concentrations (Kornberg and Baker, 1992), and its ability to enhance strand displacement synthesis could be impaired.
Figure 4: Strand displacement reactions in the presence of single-stranded binding proteins. Standard HIV-RT reactions in the presence of E. coli SSB or RP-A were carried out for 30 min as described under Methods. Only substrate B was used in this experiment. A, the concentrations of SSB in lanes 1-5 were 600, 300, 150, 75, and 37.5 nM, respectively. Assuming each tetramer of SSB covers 65 nucleotides (Kornberg and Baker, 1992), these concentrations of SSB are enough to coat 1000% to 62% of the DNA in the reaction. Lane 6 contained no SSB. B, the concentrations of RP-A used in lanes 1-4 were 35, 70, 140, and 280 nM. If each molecule of RP-A covers 30 nucleotides (Henricksen et al., 1994), these concentrations should coat 12.5, 25, 50, and 100% of the DNA in the reactions. Lane 5 contained no RP-A. The product sizes were determined using a labeled 100-bp ladder (not shown).
Finally, the effect of human RP-A on the strand displacement reactions was tested (Fig. 4B). RP-A is the human analog of E. coli SSB. It binds single-stranded DNA, and it can stimulate the activity of polymerases and helicases (Henricksen et al., 1994). Like E. coli SSB, RP-A enhanced the percent of extended primers that were longer than 400 nucleotides. Using the PhosphorImager, we measured a 4-6-fold increase in the percent of these products in the presence of 35-140 nM RP-A (lanes 1-3). When RP-A was present at 280 nM, virtually no synthesis occurred (lane 4). At 140 nM RP-A, the percent of extended primers was decreased, and, at 70 and 35 nM, the percentage of extended primers in the presence and absence of RP-A remained constant. Therefore, RP-A increases the formation of long products, even when it is present at levels high enough to decrease the total amount of synthesis. The inhibition of polymerases by RP-A has been observed previously (Tsurimoto and Stillman, 1991), possibly because high concentration RP-A blocks the binding of the polymerase to the primer terminus.
Figure 5:
Strand displacement synthesis in the
presence of an acceptor DNA. Standard HIV-RT reactions were performed
for 60 min as described under Methods. The acceptor was added
along with the MgCl and the dNTPs to start the reactions.
The first lane contains elongation of a labeled primer on the
acceptor template. The bands at position T correspond to extension
products that transferred to the acceptor template and completed
synthesis there. The bands at position 663 correspond to the
full-length extension products on the original template DNA. The
product sizes were determined using a labeled 100-bp ladder (not
shown).
Instead, the primer DNA transferred to the
acceptor DNA whether there was a downstream segment of DNA annealed to
the template or not. The primer did not have homology to the acceptor
DNA until it had been elongated by at least 10 nucleotides. Transfer in
the absence of a downstream segment of DNA, substrate A, was first
observed when the acceptor DNA was present at a 10-fold excess over the
template DNA. Transfer in the presence of a downstream segment of DNA,
substrate B, was first observed when the acceptor DNA was present at a
5-fold excess over the template DNA. Furthermore, with substrate B,
virtually no full-length products were seen. This transfer phenomenon
was not specific for HIV-RT, since Sequenase was also
found to perform this reaction (not shown).
During HIV replication, a DNA segment approximately 637 nucleotides in length needs to be displaced in order to generate the long terminal repeats. Strand displacement by HIV-RT in vitro has been documented previously for substrates 50 or less nucleotides in length (Huber et al., 1989; Hottiger et al., 1994). Preliminary results from our laboratory (not shown) using similar substrates showed a small but noticeable slowing of primer elongation in the presence of a downstream primer. This suggested that displacement of the natural LTR region, as is necessary in vivo, might be a daunting problem for the RT. When we measured strand displacement synthesis on an LTR substrate in vitro, we found that, indeed, synthesis was slow and inefficient. In fact, the primer elongation rate was reduced by approximately 12-fold from synthesis over an LTR sequence template lacking the long downstream primer. Whiting and Champoux(1994) reported that murine leukemia virus RT was able to displace 1334 base pairs of DNA with approximately a 10-fold reduction in the rate of synthesis. These results show a similarity in strand displacement rates between these two RTs despite the differences in the substrate used.
After infection by HIV-1 in cell culture, it takes 4-8 h before complete polymerization is observed (Kim et al., 1989). Synthesis of two strands of 9.5 kilobases each must occur. This requires a minimum synthetic rate of 0.65-1.31 nucleotides per s. In this study, we observed a polymerization rate of 3.3 nucleotides per s in the absence of a downstream primer. This is comparable to the polymerization rate observed by Klarmann et al.(1993) and Yu and Goodman(1992) of 1-3 nucleotides per s over heteropolymeric templates. In our system, the polymerization rate in the presence of a downstream primer dropped to 0.28 nucleotide per s.
It is still not known whether the (+)-strand is initiated at one or two sites, or if strand displacement is performed by one RT extending the (-)-DNA or by two RTs extending the(-)- and (+)-strands simultaneously (Fig. 1A). If two RTs extend the(-)- and (+)-strand simultaneously, it would take approximately the same time for one enzyme to synthesize through the whole LTR region (637 nucleotides/0.28 nucleotide/s = 38 min) as for the other enzyme to synthesize the rest of the (+)-strand (9000 nucleotides/3.3 nucleotides/s = 45 min). However, if the (+)-strand is initiated at two or more sites, strand displacement may be a rate-limiting step in the reaction. In either case, the rate of strand displacement that we have observed does not conflict with known information on the rate of viral growth in vivo. Nevertheless, it is remarkably slow compared to the synthesis rates of most DNA polymerases, which are hundreds to thousands of nucleotides per s (Kornberg and Baker, 1992).
We observed that the HIV-RT is less processive for synthesis during strand displacement. The need to frequently rebind the primer-template during synthesis contributes to the length of time needed to complete primer elongation. The RT may also have a particularly difficult time rebinding at sequences that promote pausing and are likely positions for dissociation. Klarmann et al.(1993) observed that HIV-RT preferentially initiates synthesis at unextended primers, even when pause products comprise a significant proportion of potentially available primers. This is consistent with our observation that the entire population of primers that can be elongated grow slowly throughout the reaction.
We attempted to enhance strand displacement by including single-stranded binding proteins in the reaction. Since viral NC protein normally coats part of the viral genome in vivo, we tested whether its presence would enhance the rate of synthesis. We did not see an enhancement of strand displacement synthesis over a wide range of tested NC concentrations. Current results with the LTR template showed an inhibition of DNA synthesis at high NC concentrations. We do not know why this inhibition is observed, but it does not seem to result from melting of the primer from the template by NC. On the other hand, E. coli SSB and human RP-A enhanced the extent of strand displacement in vitro. These proteins presumably act by stabilizing the displaced fragment of DNA. This makes displacement more energetically favorable. Stabilization of the single-stranded region may also discourage the partially displaced strand from promoting dissociation of either the RT or the 3` end region of the upstream primer. Hottiger et al.(1994) did not report a stimulation of the HIV-RT strand displacement reaction catalyzed by E. coli SSB. However, the effects of SSB may not have been great for the 50-nucleotide segment displaced in their system. Even though RP-A enhances strand displacement in vitro, it is unknown whether it would do so in vivo. Immunocytochemistry studies have shown that RP-A is found almost exclusively in the nucleus (Kenny et al., 1990). It is widely accepted that reverse transcription occurs in the cytoplasm (Whitcomb and Hughes, 1992). Furthermore, it has been shown that full-length(-)-strand DNA can be found in the cytoplasm (Miller et al., 1995). These findings suggest that RP-A does not influence strand displacement synthesis in vivo.
In another attempt to enhance the strand displacement reaction, we added an additional segment of single-stranded DNA to the reaction that was complementary to the downstream segment of DNA. The strand displacement/assimilation model suggests that the displaced DNA can actively migrate to a complementary piece of DNA. We expected that this process would stimulate the displacement reaction by stabilizing the displaced strand in a double-stranded form. However, the presence of this DNA strand did not stimulate the strand displacement reaction. Instead, the extending upstream primer readily transferred to the new strand sometime after it had been extended by at least 10 nucleotides. This is an interesting observation since it suggests that recombination is possible during (+)-strand synthesis. The process may be due to complex secondary structures that are formed in the LTR template. Strand transfer in vitro is promoted at positions where the RT pauses during synthesis (Wu et al., 1995), and pausing correlates with secondary structure in the template (DeStefano et al., 1992). The transfer was observed in the presence or absence of a downstream DNA, but it appeared to be more efficient in the presence of a downstream DNA primer. During displacement, when the enzyme pauses or dissociates, the already displaced segment of DNA may reanneal to the template, forcing dissociation of the 3` end region of the upstream primer. This process, called branch migration, is likely to occur between episodes of dissociation and reassociation of the RT that occur frequently during strand displacement synthesis. Once the 3` terminus is dislodged, it would be expected to associate readily with the new complementary DNA. This process is so efficient in vitro, that when the complementary DNA is present in excess, virtually all extending primers undergo transfer. We propose that this reaction is a likely means of recombination in vivo.