Delaying reverse transcription does not increase sensitivity of HIV-1 to human TRIM5α.

BACKGROUND: Because uncoating of the capsid is linked to reverse transcription, modifications that delay this process lead to the persistence in the cytoplasm of capsids susceptible to recognition by the human restriction factor TRIM5α (hTRIM5α). It is unknown, however, if increasing the time available for capsid-hTRIM5α interactions would actually render viruses more sensitive to hTRIM5α. RESULTS: Viral sensitivity to hTRIM5α was evaluated by comparing their replication in human U373-X4 cells in which hTRIM5α activity had or had not been inhibited by overexpression of human TRIM5γ. No differences were observed comparing wild-type HIV-1 and variants carrying mutations in reverse transcriptase or the central polypurine tract that delayed the completion of reverse transcription. In addition, the effect of delaying the onset of reverse transcription for several hours by treating target cells with nevirapine was evaluated using viral isolates with different sensitivities to hTRIM5α. Delaying reverse transcription led to a time-dependent loss in viral infectivity that was increased by inhibiting capsid-cyclophilin A interactions, but did not result in increased viral sensitivity to hTRIM5α, regardless of their intrinsic sensitivity to this restriction factor. CONCLUSIONS: Consistent with prior studies, the HIV-1 capsid can be targeted for destruction by hTRIM5α, but different strains display considerable variability in their sensitivity to this restriction factor. Capsids can also be lost more slowly through a TRIM5α-independent process that is accelerated when capsid-cyclophilin A interactions are inhibited, an effect that may reflect changes in the intrinsic stability of the capsid. Blocking the onset or delaying reverse transcription does not, however, increase viral sensitivity to hTRIM5α, indicating that the recognition of the capsids by hTRIM5α is completed rapidly following entry into the cytoplasm, as previously observed for the simian restriction factors TRIM-Cyp and rhesus TRIM5α.

Gag cytotoxic T lymphocyte escape mutations can increase sensitivity of HIV-1 to human TRIM5alpha, linking intrinsic and acquired immunity.

Although laboratory-adapted HIV-1 strains are largely resistant to the human restriction factor TRIM5α (hTRIM5α), we have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) responses. Recombinant viruses carrying HIV-1 CA sequences from NL4-3 and three different clinical isolates were prepared, along with variants in which mutations associated with CTL resistance were modified by site-directed mutagenesis, and the infectivities of these viruses in target cells expressing hTRIM5α and cells in which TRIM5α activity had been inhibited by overexpression of TRIM5γ were compared. For both hTRIM5α-sensitive viruses studied, CTL-associated mutations were found to be responsible for this phenotype. Both CTL resistance mutations occurring within HLA-restricted CA epitopes and compensatory mutations occurring outside CTL epitopes influenced hTRIM5α sensitivity, and mutations associated with CTL resistance selected in prior hosts can contribute to this effect. The impact of CTL resistance mutations on hTRIM5α sensitivity was context dependent, because mutations shown to be responsible for the TRIM5α-sensitive phenotype in viruses from one patient could have little or no impact on this parameter when introduced into another virus. No fixed relationship between changes in hTRIM5α sensitivity and infectivity was discernible in our studies. Taken together, these findings suggest that CTL mutations may influence HIV-1 replication by modifying both viral infectivity and sensitivity to TRIM5α.

Modulation of TRIM5alpha activity in human cells by alternatively spliced TRIM5 isoforms.

TRIM5α is a restriction factor that can block an early step in the retroviral life cycle by recognizing and causing the disassembly of incoming viral capsids, thereby preventing the completion of reverse transcription. Numerous other isoforms of human TRIM5 exist, and isoforms lacking a C-terminal SPRY domain can inhibit the activity of TRIM5α. Thus, TRIM5α activity in a given cell type could be dependent on the relative proportions of TRIM5 isoforms expressed, but little information concerning the relative expression of TRIM5 isoforms in human cells is available. In this study, we demonstrate that mRNAs coding for TRIM5α represent only 50% of total TRIM5 transcripts in human cell lines, CD4(+) T cells, and macrophages. Transcripts coding for, in order of abundance, TRIM5ι (TRIM5-iota), a previously uncharacterized isoform, TRIM5γ, TRIM5δ, and TRIM5κ are also present. Like TRIM5γ and TRIM5δ, TRIM5ι and TRIM5κ do not inhibit HIV-1 replication, but both have dominant-negative activity against TRIM5α. Specific knockdown of TRIM5ι increases TRIM5α activity in human U373-X4 cells, indicating that physiological levels of expression of truncated TRIM5 isoforms in human cells can reduce the activity of TRIM5α.

Strain-specific differences in the impact of human TRIM5alpha, different TRIM5alpha alleles, and the inhibition of capsid-cyclophilin A interactions on the infectivity of HIV-1.

HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors--capsid sequence, CypA levels, and TRIM5α--interact to determine capsid stability and therefore viral infectivity.

Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type.

BACKGROUND: HIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA) protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA) and TRIM5alpha can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A - CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5alpha by the target cells. RESULTS: Viral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5alpha expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity. CONCLUSION: Multiple interactions between host-cell factors and Gag can strongly affect HIV-1 infectivity, and these vary according to target cell type and the origin of the Gag sequence. Two of the cellular activities involved appear to be modulated in opposite directions by CypA-CA interactions, and Gag sequences determine the intrinsic sensitivity of a given virus to each of these cellular activities.

Functional central polypurine tract provides downstream protection of the human immunodeficiency virus type 1 genome from editing by APOBEC3G and APOBEC3B.

Lentiviruses utilize two polypurine tracts for initiation of plus-strand viral DNA synthesis. We have examined to what extent human immunodeficiency virus type 1 plus-strand initiation at the central polypurine tract (cPPT) could protect the viral genome from DNA editing by APOBEC3G and APOBEC3B. The presence of a functional cPPT, but not of a mutated cPPT, extensively reduced editing by both APOBEC3G and APOBEC3B of sequences downstream, but not upstream, of the cPPT, with significant protection observed as far as 400 bp downstream. Thus, in addition to other potential functions, the cPPT could help protect lentiviruses from editing by cytidine deaminases of the APOBEC family.

Detection of minority populations of HIV-1 expressing the K103N resistance mutation in patients failing nevirapine.

Salvage therapy with efavirenz is often ineffective in patients having failed nevirapine treatment, even when mutations associated with efavirenz resistance are not detected by standard population-based genotyping. The presence of minority viral populations expressing efavirenz cross-resistance could explain these observations, and such populations were sought in plasma from patients failing nevirapine for whom genotyping revealed the presence of the Y181C mutation (usually associated with limited efavirenz cross-resistance) but not the K103N mutation (which produces high-level efavirenz resistance). Viral populations expressing K103N (>1% total virus) were detected by sequence-selective polymerase chain reaction in 4 of 16 patients failing nevirapine, although, in retrospect, the mutation was not perceptible in the original genotype in only 2 cases. Both patients with detectable K103N mutations who received efavirenz failed treatment, and virus expressing K103N emerged. Four of 5 patients without detectable K103N mutations also failed efavirenz, associated with the emergence of nonnucleoside reverse transcriptase mutations that included K103N in 2 cases. The emergence of a minority viral population expressing K103N was identified in 1 patient from a separate study group subsequent to discontinuing treatment with nevirapine. These findings support the idea that minority viral populations with distinct resistance genotypes, although undetectable by standard genotyping, can contribute to the failure of salvage regimens.

Role of minority populations of human immunodeficiency virus type 1 in the evolution of viral resistance to protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) drug resistance results from the accumulation of mutations in the viral genes targeted by the drugs. These genetic changes, however, are commonly detected and monitored by techniques that only take into account the dominant population of plasma virus. Because HIV-1-infected patients harbor a complex and diverse mixture of virus populations, the mechanisms underlying the emergence and the evolution of resistance are not fully elucidated. Using techniques that allow the quantification of resistance mutations in minority virus species, we have monitored the evolution of resistance in plasma virus populations from patients failing protease inhibitor treatment. Minority populations with distinct resistance genotypes were detected in all patients throughout the evolution of resistance. The emergence of new dominant genotypes followed two possible mechanisms: (i) emergence of a new mutation in a currently dominant genotype and (ii) emergence of a new genotype derived from a minority virus species. In most cases, these population changes were associated with an increase in resistance at the expense of a reduction in replication capacity. Our findings provide a preliminary indication that minority viral species, which evolve independently of the majority virus population, can eventually become dominant populations, thereby serving as a reservoir of diversity and possibly accelerating the development of drug resistance.