Mechanistic aspects of HIV‐1 reverse transcription initiation

Repeated bottleneck passages of RNA viruses result in fitness losses due to the accumulation of deleterious mutations. In contrast, repeated transfers of large virus populations result in exponential fitness increases. Human immunodeficiency virus type 1 (HIV-1) manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells. An analysis of the mutations associated with fitness loss in four debilitated clones revealed mutation frequencies in gag that were threefold higher than those in env. We now show an increase in the fitness of the debilitated HIV-1 clones by repeated passages of large populations. An analysis of the entire genomic nucleotide sequences of these populations showed that few mutations, from two to seven per clone, mediated fitness recovery. Eight of the 20 mutations affected coding regions, mainly by the introduction of nonsynonymous mutations (75%). However, most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5 untranslated leader region of the genome, and more specifically, in the primer binding site (PBS) loop. Two of the viruses incorporated the same mutation in the primer activation signal in the PBS loop, which is critical for the tRNA 3Lys -mediated initiation of reverse transcription. Moreover, 25% of the mutations observed were reversions. This fact, together with the presence of a large proportion of nonsynonymous replacements, may disclose the operation, during large population passages, of strong positive selection for optimal HIV-1 replication, which seems to be primarily affected by binding of the tRNA to the PBS and the initiation of reverse transcription.

Section: Discussionmentioning

confidence: 99%

Few Mutations in the 5′ Leader Region Mediate Fitness Recovery of Debilitated Human Immunodeficiency Type 1 Viruses

Yuste

Bordería

Domingo

et al. 2005

“…The mechanism underlying the selection of tRNA 3 Lys by HIV-1 for use as the primer in replication is unknown. Previous studies have suggested that the viral proteins nucleocapsid and reverse transcriptase or the Gag-Pol protein are involved in selection (3,8,18,19). Recent studies have demonstrated that the lysyl-tRNA synthetase is present in HIV-1 virions and may act as a chaperone for tRNA (4).…”

Section: Discussionmentioning

confidence: 99%

Probing the Importance of tRNA Anticodon: Human Immunodeficiency Virus Type 1 (HIV-1) RNA Genome Complementarity with an HIV-1 That Selects tRNA ^Glu for Replication

Dupuy

Kelly²,

Elgavish

et al. 2003

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3′-terminal nucleotides of tRNA3Lys. Why all known strains of HIV-1 select tRNA3Lys for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNAGlu. Since the virus was not initially designed to use tRNAGlu, the virus had selected tRNAGlu from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNAGlu may provide new insights into the preference for tRNA3Lys. HIV-1 constructed with the PBS complementary to tRNAGlu was more stable than HIV-1 with the PBS complementary to tRNAMet or tRNAHis; however, all of these viruses eventually reverted back to using tRNA3Lys following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNAGlu remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA3Lys are exploited by HIV-1 for selection as the reverse transcription primer

“…In vitro studies of recombinant RT with either the W401L or W401A mutation also demonstrated a complete inhibition of RNA-dependent DNA polymerase activity using the same synthetic template/primer used for our RT assays (51) and also using a template/primer based on the HIV-1 sequence (53). Therefore, one explanation for the difference in RT activity in infected cells compared to that in vitro may be due to the stabilizing presence of other viral or host cell factors that modulate RT activity in the reverse transcription complex (17,19,54,55,63). Another possibility for the difference in RT activity of W401 mutants in cell-free assays compared to intracellular reverse transcription can be ascribed to the dimerization constant of the RT.…”

Section: Discussionmentioning

confidence: 99%

Mutations That Abrogate Human Immunodeficiency Virus Type 1 Reverse Transcriptase Dimerization Affect Maturation of the Reverse Transcriptase Heterodimer

Wapling

Moore

Sonza

et al. 2005

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerizationinhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is critical for HIV-1 replication and is required for the conversion of the genomic viral RNA into a double-stranded proviral DNA precursor, catalyzed by the RNA-and DNA-dependent polymerase and RNase H activities of the enzyme. The biologically relevant form of HIV-1 RT is a heterodimer composed of 66 (p66)-and 51-kDa (p51) polypeptides. The p51 subunit is derived from and is identical to the N-terminal polymerase domain of p66 (9). The p66 subunit can be divided structurally into the polymerase and RNase H domains, with the polymerase domain further divided into the fingers, palm, thumb, and connection subdomains (24, 29). One functional polymerase and RNase H active site is located on the p66 subunit, which adopts an "open" structure to accommodate the nucleic acid template/primer (24, 29). The p51 subunit has the same polymerase subdomains as p66. However, the spatial orientations of the individual subdomains differ from those in p66, with the p51 subunit assuming a "closed" structure and playing a largely structural role in the heterodimer (2, 23, 32). Structural analyses reveal three major contacts between the p66 and p51 subunits, which include interactions between the connection subdomains of both subunits, with most of the interaction surface being largely hydrop...