(Received for publication, June 22, 1995; and in revised form, August 9, 1995)
From the
Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3` and 5` ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5` of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5` of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5` end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.
The single-stranded plus sense RNA genome of a retrovirus is
converted into double-stranded linear DNA by the process of reverse
transcription (for review, see (1) and (2) ). At the
two ends of the viral genome, there is a direct repeat (R) sequence
that is bordered by an internal unique 5` (U5) or 3` (U3) sequence.
Immediately downstream of the 5` R-U5 sequence is the primer binding
site (PBS), ()which positions the primer tRNA used to
initiate minus strand synthesis. During reverse transcription, these
sequences are duplicated through two template jumps to generate long
terminal repeats (LTRs), which are present at both ends of the
unintegrated viral DNA and have the structure U3-R-U5 (1, 2, 3) . Upon integration into the host
cell chromosome, two base pairs are lost from the termini such that the
left LTR begins with the sequence 5`-TG . . . , and the right LTR ends
with the sequence . . . CA-3` ( (4, 5, 6) and for review, see (7) ).
The enzyme responsible for reverse transcription is the viral-encoded reverse transcriptase (RT), which contains an RNA- and DNA-dependent DNA polymerase and an RNase H activity(1, 2) . The RNase H activity serves three distinct roles during reverse transcription (for review, see (8) ). First, RNase H degrades the viral RNA genome in the RNA-DNA hybrids generated during minus strand DNA synthesis (see Fig. 1). This degradation appears to be relatively nonspecific and produces fragments ranging from 2 to 20 bases in length (9, 10, 11, 12, 13, 14, 15, 16, 17) . While concomitant DNA synthesis is not required for RNase H activity(18, 19) , the positioning of the RNase H active site is strongly influenced by binding of the polymerase to a 3` DNA primer terminus(16, 18, 20, 21) . The second role of RNase H is to cleave the viral template at the polypurine tract (PPT), a purine-rich region immediately upstream of U3, to generate the primer for plus strand DNA synthesis. Unlike genome degradation, the PPT cleavage is sequence-specific and highly precise(22, 23, 24, 25, 26) . Third, RNase H removes the RNA primers used to initiate plus and minus strand DNA synthesis. Removal of the plus strand PPT primer uniformly occurs at the junction between the RNA primer and the first deoxynucleotide of plus strand DNA (25, 26) . In contrast, removal of the tRNA primer from minus strand DNA appears to depend on the type of virus examined. For avian myeloblastosis virus, the primer is cleaved precisely between the last ribonucleotide of the tRNA and the first deoxynucleotide of the minus strand(27, 28) . However, the tRNA primer of human immunodeficiency virus type 1 (HIV-1) is cleaved between the last and the penultimate ribonucleotides to leave a ribo A residue at the 5` end of the minus strand DNA(18, 29, 30) .
Figure 1: RNase H cleavage sites important for second jump in reverse transcription. The current model of reverse transcription is depicted in sequential steps (stages i-v) through the second template jump, with RNA strands as light lines and DNA strands as dark lines. Viral sequences that serve as landmarks for replication are shown in capital letters, and the complementary sequences are indicated with a prime. The 3` end of each strand is indicated by an arrowhead. For the second jump, RNase H cleavages are required in the regions marked I and II. RNase H cleavage of the genome template in region I (stage i) determines the maximal position to which the minus strand can be extended prior to the second jump (stage iv). RNase H cleavage in region II (stage iii) removes the tRNA primer to free the PBS sequences for the second jump and determines the 5` end of the minus strand (stage iv). The second jump intermediate is depicted in stage v.
The model of reverse transcription predicts that RNase H activity is necessary for both the first and second template jumps to occur (3) (see Fig. 1). After minus strand synthesis has extended the tRNA primer to the end of the genome (minus strong stop DNA; stage i), RNase H facilitates the first jump by degrading some portion of R at the 5` end of the viral RNA. This frees minus strand R` for transfer to R at the 3` end of the genome and continuation of synthesis (stage ii). Consistent with this model, RNase H cleaves the template RNA up to 14-24 nucleotides from its 5` end in DNA-RNA hybrids, including those which contain R(20, 31, 32, 33) . While some in vitro studies using model substrates and RNase H-deficient RT have found that RNase H activity is necessary for a template switch(15, 34) , others have reported that RNase H activity is not required(32, 35) . However, viral mutants lacking RNase H activity do not complete reverse transcription in vivo and only synthesize minus strong stop DNA in endogenous reactions (36).
For the second jump, cleavages by RNase H are important for three different stages of reverse transcription (see Fig. 1). The first cleavage occurs on the genome 5` of the PBS after minus strand synthesis has begun (designated ``region I,'' stage i). The 3`-most cleavage in this region determines the maximum point to which the minus strand DNA can be extended prior to the second jump (stage iv). Whether this RNase H cleavage is random or highly specific has not been previously addressed. The second RNase H cleavage removes the tRNA primer after plus strand synthesis has extended from the PPT primer through to the end of PBS (designated ``region II,'' stage iii). This cleavage determines the 5` end of the minus strand and presumably frees the 3` end of plus strand DNA for the second jump (stage iv). Finally, the PBS` sequence of the extended minus strand DNA is exposed by RNase H degradation of the genomic template (stage iv). Together, these RNase H cleavages permit the PBS sequence of the plus strand to pair with the PBS` sequence of the minus strand for the second jump (stage v). The specificity of RNase H for the first two cleavages is an intriguing issue, as any duplication in sequence between the 3` and 5` ends of minus strand DNA could generate a problematic branched intermediate subsequent to the second jump (stage v).
Using Moloney murine leukemia virus (M-MuLV) as a model system, we have examined the specificities of RNase H that define the two termini of minus strand DNA prior to the second jump. These results allow us to predict the predominant structure that would result from the second jump according to the current model of reverse transcription.
Oligonucleotides were 5` end-labeled in a 15-µl reaction
containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl, 5 mM dithiothreitol (DTT), 50 µg/ml
of bovine serum albumin, 50 µCi of
[
-
P]ATP (DuPont NEN), 17 pmol of
oligonucleotide, and 2-30 units of T4 polynucleotide kinase. The
reaction was incubated at 37 °C for 60 min and stopped with
10-50 mM EDTA. In most cases, the labeled
oligonucleotides were separated from labeled ATP using a spin column
containing Sephadex G-25 (Pharmacia Biotech Inc.)(38) .
Figure 2:
Assays for RNase H cleavage sites. The
drawings show the different assays used to map RNase H cleavages in the
genome 5` of the PBS (A) and the RNase H cleavage event that
removes the tRNA primer (B). RNA strands are depicted as light lines, DNA strands as dark lines, and relevant
viral sequences in capital letters. The asterisks indicate P-labeled 5` ends of RNA or DNA
oligonucleotides as specified in the text, and the 3` end of each
strand is indicated by an arrowhead. Depending on the
experiment, the RNA paired with the PBS (PBS`) is either the
RNA oligo Mol15R or the primer tRNA. A, primer extension from
labeled oligo 1 maps cleavages on the plus strand genome RNA. B, extension from labeled oligo 2 was used to map the 5`-end
of the minus strand DNA after primer removal and 5`-end-labeled Mol15R
was used to determine the fate of the RNA
primer.
Figure 3:
Determination of the cleavage sites within
the plus strand U5-PBS region. RNA oligo Mol15R was annealed to a
template RNA containing R-U5-PBS sequences and either used directly as
a substrate for RT (Primer only) or Mol15R was extended with
RNase H RT (Superscript II) prior to use as a
substrate (Extended primer) (see Fig. 2A). The
5` ends of the template RNAs were mapped either before (lanes 1 and 3) or after treatment with recombinant M-MuLV RT for
15 min (lanes 2 and 4) by primer extension using
labeled oligo 1. Primer extension products were analyzed in an 8%
polyacrylamide, 8 M urea gel adjacent to a dideoxy-sequencing
ladder generated using the same labeled oligonucleotide (lanes
T, G, C, and A). The lower signal in lane 2 resulted from cleavages in the RNA 3` of the PBS due to
the presence of some snap-back priming during the preparation of the
substrate (see ``Materials and Methods''). Arrow a indicates the mobility of products that extend to the 5` end of
the RNA template; arrow b marks the predominant extension
product that results from the RNase H cleavage site within the hybrid
region just 5` of the PBS; and arrow c indicates the position
of products resulting from cleavage within the PBS. The RNA sequence of
the U5-PBS region is shown with the PBS boxed at the left.
We next examined the RNase H cleavage pattern on genomic RNA upstream of the PBS in endogenous reactions. Detergent-disrupted M-MuLV virions were incubated with dNTPs, and products synthesized over time were analyzed by primer extension using oligo 1 (Fig. 2A). In the absence of synthesis (zero time), the primer extension products were similar to those observed with substrates containing unextended RNA in the in vitro assay with model substrates (compare Fig. 4, lane 1, with Fig. 3, lane 4). The vast majority of products mapped to the 5` end of the genomic RNA (Fig. 4, lane 1, arrow a), with a small fraction mapping to the PBS region itself (arrow c). Cleavages within the PBS could have resulted from very low levels of RNase H* activity within the virions prior to DNA synthesis. It would appear that these minor cleavage products persisted throughout the time course, although the bands are fainter in the 4-24 h time points (Fig. 4, lanes 2-5) owing to a reduced recovery of products for these samples. After 4 h of synthesis, the amount of genomic length products decreased dramatically, and several products mapping immediately 5` of the PBS appeared. The major product was identical to that observed using the RNA-DNA hybrid substrate in the in vitro assay and corresponded to RNase H cleaving the viral genome between the second and third ribonucleotides 5` of the PBS (Fig. 4, lane 2, arrow b). Several minor cleavages were also detected, beginning with the first ribonucleotide 5` of the PBS and extending up to 10 ribonucleotides 5` of the PBS. The relative abundance of these secondary cleavage sites decreased with increasing lengths of time, reflecting further RNase H activity on the genomic RNA template as reverse transcription progressed (Fig. 4, lanes 3-5).
Figure 4: Cleavage sites on genomic RNA after minus strand synthesis in the endogenous reaction. Using detergent-disrupted M-MuLV virions, endogenous reverse transcription reactions were carried out for 4 h (lane 2), 8 h (lane 3), 16 h (lane 4), or 24 h (lane 5), and products were recovered by phenol extraction and ethanol precipitation. Products were similarly prepared from detergent-disrupted virions in the absence of any synthesis (0 h; lane 1). To map the 5` ends of genomic RNA upstream of the PBS, primer extensions were performed with labeled oligo 1, and samples were analyzed as described in Fig. 3. The designations for the arrows and RNA sequence are identical to Fig. 3.
Figure 5:
Determination of the 5` end of minus
strand DNA at the U5`-PBS` junction after primer removal. A,
RNA oligo Mol15R was annealed to single-stranded EcoRI-cut
M13mp7/PBS DNA and extended with Sequenase (see Fig. 2B). The resulting substrate was treated with
recombinant M-MuLV RT for 10 min (lanes 3, 4, 7, and 8) or not treated (lanes 1, 2, 5, and 6), and the 5` ends of the minus
strand DNAs were mapped by primer extension using labeled oligo 2A and
RNase H
RT (Superscript) (lanes 1-4)
or T4 DNA polymerase (lanes 5-8). To remove any
remaining RNA oligonucleotide from the 5` ends of the minus strands,
the products in the indicated lanes were treated with alkali before
carrying out the primer extension reactions. Primer extension products
were analyzed adjacent to a dideoxy sequencing ladder generated using
labeled oligo 2A (lanes T, G, C, and A) as described in Fig. 3. B, same as for A except that the substrate was not treated (0 min, lanes
1 and 2) or treated with recombinant RT for 1 min (lane 3), 3 min (lane 4), 10 min (lane 5),
30 min (lane 6), or 60 min (lane 7), and primer
extensions were performed only with RNase H
RT. For
each panel, the sequence of the RNA oligonucleotide is indicated at the left. Arrow a marks the position of extension
products corresponding to the 5` end of the RNA primer; arrow b indicates the position of the major cleavage product containing
one ribonucleotide A on the 5` end of the minus strand; and arrow c indicates the position of the 5` end of the
DNA.
Since a portion of the molecules was not cleaved by RNase H in this experiment (Fig. 5A, lane 4, arrow a), we tested whether longer reaction times might facilitate removal of the ribo A. As shown in Fig. 5B, treatment with RT through 60 min greatly reduced the amount of 5` ends containing the full-length RNA primer (arrow a, lanes 3-7). Notably, 5` ends containing a single ribo A increased (arrow b), and in other experiments, the ribo A persisted after incubating the substrates with RT for as long as 120 min (data not shown).
We next addressed whether primer removal by RNase H
during the endogenous reaction also leaves a ribo A at the 5` end of
minus strand DNA. The minus strand products of a 16-h endogenous
reaction were isolated and analyzed by primer extension reactions using
oligo 2 before and after alkali treatment to determine whether any
ribonucleotides remained on the 5` end of the DNA. Pretreatment with
alkali yielded the expected T4 DNA polymerase primer extension product
corresponding to the 5` end of the minus strand DNA (Fig. 6, lane 3). When the same alkali-treated sample was analyzed
using RNase H RT as the primer-extension polymerase,
a second band one nucleotide shorter than the expected product was
observed (Fig. 6, lane 2). Since only a single product
was observed with T4 DNA polymerase, we conclude that the shorter
primer-extension product observed in lane 2 resulted from
RNase H
RT pausing one nucleotide short of the 5` end
of the template. It is unclear why pausing at this site was
consistently more pronounced with the products of the endogenous
reaction as compared with those of the reconstituted reaction (see Fig. 5A, lane 4). The primer extension
analysis on the sample that had not been treated with alkali revealed
three bands in addition to the expected full-length product resulting
from complete extension through the RNA primer (Fig. 6, lane
1). The upper of these three bands corresponds to a ribo A on the
end of a significant fraction of the minus strands initiated in the
endogenous reaction since this product disappears after alkali
treatment (compare lanes 1 and 2). The middle band
corresponds to cleavage precisely at the RNA-DNA junction, while the
fastest migrating species results from premature termination by RNase
H
RT during the primer extension assay. Because
premature termination could, in principle, occur on molecules either
with or without the ribo A residue, it is not possible to accurately
quantify the proportion of molecules in each class from the endogenous
reaction. Since T4 DNA polymerase did not efficiently copy
ribonucleotides in primer extensions carried out on the model
substrates (see Fig. 5A, lanes 6 and 8), this enzyme could not be used to confirm that the
extension product, which is two nucleotides shorter than ribo A species
in Fig. 6, lane 1, indeed resulted from premature
termination.
Figure 6:
Determination of the 5` end of minus
strand DNA synthesized in the endogenous reaction. An endogenous
reaction was incubated for 16 h, and the 5` ends of the minus strand
DNAs were mapped using labeled oligo 2A and RNase H RT (Superscript) (lanes 1 and 2) or T4 DNA
polymerase (lane 3) as described in Fig. 5. Samples
shown in lanes 2 and 3 were treated with alkali. The
sequence of the tRNA primer is indicated at the left.
To ask whether the recombinant RT differs from the
virion-derived RT in its ability to cleave at the RNA-DNA junction, we
directly compared the RNase H activities of these enzymes on the model
substrate described in Fig. 2B. The substrate was
incubated with either RT purified from M-MuLV virions or the
recombinant RT for 10 or 90 min, and the products were analyzed by
primer extension using labeled oligo 2 and RNase H RT. For both enzymes, a ribo A remained at the 5` end of minus
strand DNA (Fig. 7, lanes 4, 6, 8,
and 10). Treatment of identical samples with alkali prior to
the primer extension analysis confirmed the presence of the ribo A on
the 5` ends of the DNAs (Fig. 7, lanes 3, 5, 7, and 9). Based on these results, it would appear
that the virion-derived RT is indistinguishable from the recombinant
enzyme.
Figure 7:
Primer removal by RT purified from
virions. Substrates were prepared as described in Fig. 5and
treated with purified virion M-MuLV RT (Virion RT; lanes
3-6) or recombinant RT (Recomb. RT; lanes 7-10) for 10 min (lanes 3, 4, 7, and 8) or 90 min (lanes 5, 6, 9, and 10). Untreated samples are shown in lanes
1 and 2. Samples in lanes 1, 3, 5, 7, and 9 were treated with alkali prior
to the primer extension analysis by RNase H RT using
oligo 2B. The sequence of the RNA oligonucleotide is indicated at the left.
Although retroviral RNase H activity is maximal in the absence of KCl, typical RT conditions contain 40-50 mM KCl(31, 41) . We have varied the concentration of KCl with the model substrates and endogenous reactions to find that, under all conditions, the ribo A remained at the 5` end of minus sense DNA (data not shown).
Figure 8:
Time course of RNA primer cleavage by
RNase H. 5` end-labeled RNA oligo Mol15R was annealed to
single-stranded EcoRI-cut M13mp7/PBS DNA and
extended with Sequenase (see Fig. 2B). The resulting
substrate was gel-purified, treated with recombinant RT for 0.3 min (lane 3), 1 min (lane 4), 3 min (lane 5), 9
min (lane 6), or 27 min (lane 7) and analyzed in a
20% polyacrylamide, 8 M urea gel. As controls, the untreated
substrate (lane 2, arrow a) and 15-mer RNA oligo
Mol15R without extension (lane 1, arrow b) are shown.
Positions of size markers generated by nuclease P1 digestion of labeled
Mol15R are indicated at the right.
Figure 9: Removal of the RNA primer by M-MuLV versus HIV-1 RT. Substrates prepared as described in Fig. 8were treated with recombinant M-MuLV (lanes 2-4) or purified HIV-1 (lanes 6-8) RT for 0.3 min (lanes 2 and 6), 3 min (lanes 3 and 7), or 27 min (lanes 4 and 8) and analyzed as described in Fig. 8. Substrates without treatment (0 min; lanes 1 and 5) and the labeled RNA oligonucleotide without extension (lane 9) are also shown. Positions of size markers are shown on the right as described in Fig. 8.
To determine whether such a structure would affect completion of the minus strand, we used a model system designed to compare initiation and early synthesis on templates representing either the branched or nicked product of the second jump. Oligonucleotides corresponding to the 5` end (oligo U5mis) and two possible 3` ends of the minus strand were annealed to single-stranded DNA containing the plus sense LTR (Fig. 10A). Oligo U5mis contained 5 unpaired 3`-terminal nucleotides to prevent its utilization as a primer, and the two 3` upstream oligonucleotides contained either the minus sense PBS sequence (oligo PB) or the extended PBS sequence (oligo PBA) to generate a nicked or three base-branched substrate, respectively. Shown in Fig. 10B is a graph depicting the time course of displacement of the 5` end-labeled downstream oligonucleotide (oligo U5mis) as determined by nondenaturing polyacrylamide gel electrophoresis. Of note is that RT utilized and extended oligo PBA nearly as well as fully base-paired oligo PB, although a slight but reproducible lag in displacement and an apparent decrease in the total amount of displaced product were associated with the branched substrate. In a separate experiment, it was found that incubation of the templates with RT for 3 min prior to the initiation of synthesis with dNTPs did not decrease the observed lag (data not shown).
Figure 10:
Displacement synthesis on model
primer-templates comparing an unpaired with a paired primer terminus. A, schematic diagram showing the structures of the model
primer-templates constructed from a tandem pair of DNA oligonucleotides
corresponding to the putative 5` and 3` ends of the completed minus
strand annealed to plus sense LTR-containing single-stranded DNA. The arrow indicates the primer terminus that in structure I is base paired with the template and in II is unpaired
and extends beyond the 5` end of the downstream oligonucleotide by
three deoxyadenosine residues. The 3` terminus of the downstream
oligonucleotide is not complementary so it cannot be used as a primer,
and the 5` end is P-labeled (indicated with an asterisk). B, The release of the labeled
oligonucleotide by RT-mediated displacement synthesis is shown as a
function of time for the two primer-template
combinations.
We have examined the RNase H cleavages that determine the length of minus strand DNA prior to the second jump during reverse transcription of M-MuLV. In both the endogenous reaction and on a hybrid RNA-DNA substrate, RNase H cleaved the genomic template in U5 very proximal to the PBS. The primary RNase H cleavage site was between the second and third ribonucleotides 5` of the PBS (Fig. 11A), but other cleavages extending through 10 nucleotides 5` of the PBS were also apparent. These cleavages define the 5` end of the template for minus strand synthesis and would allow the majority of the minus strands to extend only a few nucleotides beyond the PBS prior to the second jump (Fig. 11B). Interestingly, Götte et al.(42) have shown that HIV-1 RT also cleaves the RNA genome two bases upstream from the U5/PBS boundary during minus strand synthesis. Using model substrates, we also found that RNase H cleaves the tRNA primer between the last and penultimate ribonucleotides (Fig. 11C), leaving a single ribo A residue at the 5` end of minus strand DNA (Fig. 11D). Similar results were obtained from an analysis of the products of the endogenous reaction except in this case some of the molecules were apparently cleaved instead at the RNA-DNA junction. We cannot rule out the possibility that a ribonuclease other than RNase H was responsible for the cleavage at the DNA-RNA junction or that this difference results from a coupling of the cleavage to concomitant DNA synthesis. Subsequent to the initial cleavage, RNase H cleaved within the RNA primer to generate smaller fragments.
Figure 11: RNase H cleavages that generate the two ends of the minus DNA strand and the resulting structure after the second jump. The sequences at the U5-PBS border are indicated for a number of structures synthesized during reverse transcription with the RNA sequences shaded. A, the arrow above the RNA sequence indicates the major RNase H cleavage site 5` of the PBS that determines the 5` end of the template for minus strand DNA synthesis. B, predicted 3` sequence for the major minus strand product assuming synthesis proceeds to the end of the template diagrammed in A. C, the arrow indicates the site of the RNase H cleavage that removes the tRNA primer and determines the 5` end of the minus DNA strand shown in D. E, the predominant structure that is predicted after the second jump. Note that the three unpaired A residues, which extend from the site of the nick, can become paired with the opposing plus strand to varying extents by branch migration (see text).
The apparent 5` and 3` termini for M-MuLV minus strand DNA suggest a detailed structure for the intermediate after the second jump of reverse transcription (Fig. 11). Upon initiation at the polypurine tract, plus strand DNA synthesis extends through the PBS and terminates at the first modified base in the tRNA(43) . To continue synthesis, plus strand strong-stop DNA transfers to the 3` terminus of minus strand DNA (Fig. 1, stage v), with the complementary sequences (PBS/PBS`) at the ends of minus strand DNA facilitating the jump(3) . We predict that the first three bases at the 3` end of the minus strand (3`-AAA . . . 5`) are not initially base-paired to the plus strand DNA (Fig. 11E). As a consequence, branch migration is necessary to position the free 3` end of the minus strand for use as a primer, and then displacement synthesis is required for minus strand elongation. In contrast, the 3` end of plus strand DNA is situated for immediate extension after the second jump. Only the intermediate arising from the major RNase H cleavage between the second and third nucleotides upstream of PBS is shown in Fig. 11E, but others are also predicted by our results. These additional structures would possess unpaired 3` termini, which are even longer, resulting in a greater requirement for branch migration prior to the initiation of displacement synthesis.
For the intermediate structure proposed for the second jump, we inferred the structure of the 3` end of the minus strand by assuming that synthesis proceeds to the 5` end of the RNA template prior to the jump. Although we cannot rigorously exclude that the second jump occurs before completion of the minus strand, this possibility seems unlikely for the following reasons. First, given the displacement activity of RT (44) , there is no compelling reason to suppose that the known secondary structure in the template in the U5-PBS region (for review, see (45) ) would present a barrier to the completion of the minus strand. Second, it seems unlikely that strand transfer would precede the extension of minus strand DNA through to the end of the RNA template because degradation of the template by RNase H is required for the first jump (36) and is likely to be required for the second jump as well(3) . Based on footprinting and structural studies, the DNA polymerase and RNase H active sites of RT are separated by approximately 18 base pairs(46, 47, 48) . Since the PBS of M-MuLV is 18 nucleotides in length, the RT that is synthesizing the minus strand would be near the 5` end of the genomic template before the RNase H activity would be positioned for cleavages in the PBS. The properties of partial deletion and substitution mutants in the HIV-1 PBS further suggest that minus strand synthesis may typically traverse the entire PBS prior to the second jump(49, 50) . Notably, Luo and Taylor (32) found that 20 but not 10 nucleotides are sufficient for the M-MuLV strand transfer reaction in vitro.
An interesting question is whether the proposed branched intermediate created by the second jump slows minus strand DNA synthesis significantly (Fig. 11E). Extension of minus strands subsequent to the second jump would require resolution of the unpaired primer by branch migration, which in principle could be facilitated by RT. If such branch migration were to be an impediment to extension of the minus strand, resolution could result from displacement through the LTR from the 5` end by extension of the plus strand DNA. Using substrates that model the predicted second jump product, RT extended a 3` end requiring a three-base branch migration nearly as efficiently as a completely base-paired 3` primer terminus. The slight lag in extension associated with the unpaired primer terminus suggests that the structure was not resolved by branch migration during the 5-min annealing period prior to the initiation of synthesis. Additionally, the failure of RT to reduce the lag following an incubation with the template minus dNTPs suggests that, at least under nonsynthesis conditions, RT does not alter the equilibrium between the base paired and unpaired forms. Given that reverse transcription occurs over a period of hours(1) , these results suggest that, although an unpaired 3` terminus is not without consequence for RT, the magnitude of the delay is small enough that it is unlikely to have a significant impact on the overall course of retroviral replication. It is uncertain what effect longer extensions of the minus strand 3` end would have on minus strand synthesis following the second jump. We would anticipate that an increase in the size of the unpaired primer terminus would lead to an increase in the lag before synthesis and perhaps a decrease in the overall utilization of the 3` minus strand following the second jump.
The presence of a ribo A at the 5` terminus of the majority of M-MuLV minus strands may affect the structure of the right LTR end and is interesting to consider with regard to integration. During retroviral integration, two base pairs are generally lost from each end of the linear viral DNA such that the first and last two bases of the integrated virus are highly conserved and begin with 5`-TG . . . at the left terminus and end with . . . CA-3` at the right terminus (for review, see (7) ). The avian RT precisely cleaves its tRNA primer at the junction between minus strand DNA and RNA(27) , which generates symmetrical ends for integration. In contrast, the tRNA primer of HIV-1 is incompletely removed, and a ribo A remains at the 5` end of the minus strand on model substrates(18, 29, 30) . These biochemical studies are supported by the observation that most HIV-1 circle junctions contain an additional A, which is not present in the predicted sequence (51, 52, 53) and that viral double-stranded DNA contains the extra base pair in the right LTR(54) . However, the presence of a ribo A at the 5` end of the HIV-1 minus strand DNA still results in symmetrical LTRs from which two base pairs are removed from both ends during integration.
In the case of M-MuLV, if the ribo A residue is retained on the 5` end of minus strands, and synthesis of plus strands extends to the end of the minus strand template to copy the ribo A, then the right end of linear unintegrated DNA would end with the sequence 5` . . . CATTT-3`, and three base pairs would have to be removed from the right LTR end during integration. It seems unlikely that the additional base pair would affect viral integration as previous studies have demonstrated that extra sequences flanking the conserved 5`-TG . . . and . . . CA-3` bases at the termini of M-MuLV do not interfere with integration(55, 56) . The addition of two extra base pairs to the right LTR resulted in a viral mutant that replicated and retained the two base pair addition. Interestingly, two of the four circle junction sequences analyzed for this virus contained an additional T that might have arisen from incomplete removal of the tRNA(56) .
Alternatively, M-MuLV minus strand DNA may undergo some additional processing that specifically removes the ribo A prior to the completion of plus strand synthesis or integration. Although we did not observe removal of the ribo A residue in the reconstructed reactions in vitro, it appeared that the ribo A was removed from some minus sense DNA ends in the endogenous reaction. Thus we might expect a mixed population of molecules with respect to the structure of the 5` ends of minus strands in vivo. The sequence of the one reported M-MuLV circle junction (57) does not contain an extra A at the position predicted by our data, but given the small sample size, it is difficult to generalize from this observation. Several studies examining the M-MuLV double-stranded DNA product of reverse transcription failed to detect minus strand DNA with a length suggesting the presence of an extra A at the 5` end(43, 58, 59) , and one of these studies did not find an extra T at the 3` end of plus strand DNA(43) . While these observations might indicate that the ends of intracellular intermediates are different from those generated in this study, it remains equally likely that the ribo A is somehow removed from minus strand 5` ends after the second jump but before completion of the plus strand. Sequence analyses of additional M-MuLV circle junctions might reveal the relative frequency of occurrence of the two predicted structures for the 5` ends of minus strands.
It remains to be
determined why the RNase H activities of both M-MuLV and HIV-1 can
remove the plus strand primer precisely at the RNA-DNA junction (25, 26) yet prefer to cleave the tRNA primer one
nucleotide away from the junction to leave a ribonucleotide on the DNA
strand. Interestingly, Huang et al.(60) showed that
the calf thymus RNase H1 invariably cleaves one base away from the
RNA-DNA junction. Through the use of two-dimensional NMR studies, the
structure of a duplex oligonucleotide that models the M-MuLV tRNA-minus
DNA junction has been determined. ()Structural anomalies at
the RNA-DNA junction, which include distortions in the base pairs, a
bend in the helix axis, and unusual sugar conformations could influence
the cleavage specificity of RNase H. Recently, it was suggested that
the width of the minor groove could be an important feature recognized
by RNase H(61) . Based on the NMR analysis of the Moloney
structure, it is possible that narrowing effects of the junctional DNA
base pair on the minor groove could preclude cleavage by RNase H at the
junction. The determination of which, if any, of these structural
features prevents removal of the ribo A by the viral RNase H awaits a
structural analysis of a substrate that is cleaved precisely at the
RNA-DNA junction.