Initiation of RNA‐dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV‐1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV‐1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo‐ and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV‐1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV‐1 RNA, HIV‐1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer‐template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.

Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO or by the set of proteins UL128, UL130, and UL131, forming gH/ gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts but fail to enter epithelial/endothelial cells, and this has led to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that strains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type-specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with that of gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131 but low in gH/gL/gO could be overcome by treatment with the chemical fusogen polyethylene glycol (PEG), strongly arguing that gH/gL/gO provides the conserved herpesvirus gH/gL entry function of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types. IMPORTANCEThe functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell-type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies and underscore the importance of gH/ gL/gO as another important part of vaccine or therapeutic strategies. P rimary infection of healthy adults by human cytomegalovirus (HCMV) is usually subclinical or mildly symptomatic but leads to lifelong persistent or latent infection. Primary infection or reactivation of HCMV in immunocompromised hosts, such those infected with HIV and t...

We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV‐1) RNA and HIV‐1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV‐1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV‐1 RT are not very different for the two complexes, RT dissociates approximately 200‐fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre‐formed primer‐template‐RT complexes is reduced by a approximately 50‐fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV‐1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post‐transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA 3 Lys , and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primertemplate complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2-hydroxyl group at the 3 end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA 3Lys . The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA 3 Lys . The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix ␣H in the p66 thumb subdomain and the RNase H domain of RT, respectively.Reverse transcription is the central event of the retrovirus life cycle (1-3). Reverse transcriptase (RT) 1 , the viral enzyme that converts the homodimer of single-stranded genomic RNA into double-stranded DNA, is a multifunctional enzyme that possesses RNA-and DNA-dependent DNA polymerase activities, as well as an RNase H function (reviewed in Ref. 3). In addition, complete DNA synthesis requires two strand transfers, which are also mediated by RT (4 -6).Like most replicative DNA polymerases, RT uses RNA primers to initiate DNA synthesis. RT initiates synthesis of the (Ϫ) strand DNA from the 3Ј end of a specific cellular tRNA species that is complementary to the primer binding site (PBS) of the genomic RNA (reviewed in Refs. 7-9). The primer for (ϩ) strand DNA synthesis is a polypurine tract that is resistant to the RNase H activity of RT (10). Evidence for specific initiation of (Ϫ) strand DNA synthesis in retroviruses and retrotransposons has recently accumulated. Complex species-specific interactions have been identified in vitro between the primer tRNA and genomic RNA of avian retroviruses (11, 12), human immunodeficiency virus type 1 (HIV-1) (13,14), and the yeast retrotransposon Ty1 (15). In HIV-1, in addition to the interaction of the 18 3Ј-terminal nucleotides of the replication primer tRNA 3 Lys with the PBS, the anticodon loop, the 3Ј strand of the anticodon stem, and part of the variable loop of the tRNA interact with the genomic RNA (13, 14, 16). The interaction between the tRNA anticodon loop and HIV-1 RNA is required for optimal replication and reverse transcription in infected cells (17)(18)(19)(20). Post-transcriptional modifications of tRNA 3 Lys are required for stabilizing the primer-template interactions (13,14) and for eff...