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Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance., Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.

We investigated the effect of N348I alone and with M184V on nonnucleoside reverse transcriptase inhibitor (NNRTI) drug susceptibility and replicative capacity in B and non-B HIV-1 isolates. N348I reduced the susceptibility to all NNRTI drugs across subtypes. The replication capacity of all viruses in a variety of cell lines was impaired by N348I. Interestingly, the N348I and M184V double mutation compensated for the reduced NNRTI drug susceptibility observed in the N348I single mutant and marginally improved viral replicative capacity.

TRIM5α proteins recruit and restrict incoming cytoplasmic retroviruses. Primate TRIM5α sequence diversity underlies species-specific restriction and is likely caused by selective pressure from ancient pathogenic infections. Here we show that TRIM5α from the European brown hare restricts diverse retroviruses. Furthermore, it differs significantly in sequence from TRIM5α from the closely related rabbit, suggesting evolutionary changes in the last 12 million years since these species diverged. We propose that, like primates, lagomorphs have been subject to selective pressure from TRIM5-sensitive viruses, possibly related to the endogenous lentivirus RELIK found in both rabbits and hares.

Mutations can accumulate in the protease and gag genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in gag have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length gag , protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length gag , protease, and gag -protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant gag gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant gag gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of gag from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant gag alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of gag sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.

Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration.

TRIM5α is a potent barrier to cross-species retroviral transmission, and TRIM5αs from different species have divergent antiretroviral specificities. Multiple TRIM5 alleles circulate within rhesus macaque populations. Here we show that they too have different antiretroviral specificities, highlighting how TRIM5 genotypes contribute to protection in an individual or a population.

Murine primary cells are poorly permissive to human immunodeficiency virus type 1 (HIV-1) vector infection. Retroviral infectivity is influenced by dominant inhibitors such as TRIM5α. Sensitivity to TRIM5α is altered by interactions between cyclophilin A and the HIV-1 capsid. Here we demonstrate that competitive inhibitors of cyclophilins, cyclosporine or the related Debio-025, stimulate HIV-1 vector transduction of primary murine cells, including bone marrow and macrophages, up to 20-fold. Unexpectedly, the infectivity of an HIV-1 mutant or a simian lentivirus that does not recruit cyclophilin A is also stimulated by these drugs. We propose that cyclosporine and related compounds will be useful tools for experimental infection of murine primary cells. It is possible that HIV-1 infection of murine cells is inhibited by dominant factors related to immunophilins.

TRIM5α is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5α antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5α in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5α-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5α expression, and expression of simian TRIM5α in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5α and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5α, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5α. We propose that cyclophilin A isomerization of a proline residue in the TRIM5α sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5α. In humans, where HIV-1 has adapted to bypass TRIM5α activity, the effects of cyclosporine A are independent of TRIM5α. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5α-independent innate immune pathway in human cells.

Selective pressure imposed by retroviral infection has driven the evolution of antiviral cellular factors that contribute to defense mechanisms against retroviruses. Products of the tripartite motif 5 (TRIM5) gene in primates and the Friend virus susceptibility factor-1 (Fv1) gene in mice constitute a class of restriction factors that inhibit retroviral infection, targeting incoming viral capsids and preventing the establishment of a provirus (6, 12, 14, 17, 20, 23, 26, 31). Restriction of viral infectivity by such factors determines retroviral tropism at the species level, and zoonotic viral transfer between species is likely to require insensitivity to these antiviral mechanisms. Fv1 was first described as one of a series of loci controlling mouse susceptibility to leukemia induced by the Friend strain of murine leukemia virus (MLV) (17). Two main alleles of Fv1 have been described, Fv1n from NIH mice and Fv1b from BALB/c mice. Fv1 enabled division of MLVs into subgroups. N-tropic MLV (MLV-N) strains are able to infect Fv1n/n cells (or NIH mice) but not Fv1b/b cells (or BALB/c mice), whereas B-tropic MLV strains (MLV-B) display the opposite phenotype, infecting Fv1b/b cells (or BALB/c mice) but not Fv1n/n cells (or NIH mice) (24). NB-tropic strains (MLV-NB), which include Moloney MLV, constitute a third Fv1 sensitivity phenotype able to infect cells of any Fv1 genotype. Fv1 blocks MLV infection in a saturable way after the virus has entered target cells and after viral reverse transcription but before the establishment of a provirus (13). The target of Fv1 restriction is the viral capsid protein, as sensitivity to Fv1 depends specifically on residues within the capsid (CA). The mechanism by which Fv1 blocks retroviral infection and the point in the viral life cycle at which Fv1 recognizes capsid remain obscure. Humans lack an Fv1 orthologue yet display an Fv1-like activity against MLV-N (28). The human gene encoding this activity was originally called Ref1 and is now known as TRIM5α (12, 14, 20, 31). TRIM5α is a member of the tripartite motif (TRIM) protein family that is defined by a cluster of three motifs with a characteristic ordering and spacing: an N-terminal RING domain, one or two B-Box type 1 or type 2 domains, and a coiled-coil region (21). Notably, sensitivity to both Fv1b and human TRIM5α (huTRIM5α) is determined by CA residue 110, where arginine denotes N tropism and glutamate B tropism (15, 28). Thus, CA mutation R110Q in N-tropic AKV WN41 leads to complete insensitivity to both Fv1b and huTRIM5α. Residues around this site, however, are also important in determining sensitivity to restriction, particularly for the attainment of an Fv1-insensitive NB phenotype (16, 25). Moreover, certain mutations are able to distinguish between Fv1 and huTRIM5α sensitivity, for example, CA D92E mutation in MLV Friend strain renders it sensitive to Fv1b but not to human TRIM5α (16). Mutational analysis of TRIM5α has contributed to a model for restriction. Swapping SPRY domains between TRIM5α alleles or SPRY domain mutagenesis demonstrates that it encodes the antiviral specificity determinant (18, 19, 22, 27, 32). The importance of the SPRY domain is also illustrated by the owl monkey example, where the TRIM5 SPRY domain has been replaced by an in-frame cyclophilin A pseudogene (23). The CypA domain recruits the TRIM5 RBCC domain to incoming human immunodeficiency virus type 1 (HIV-1) capsids, leading to restriction of HIV-1 infectivity. The molecular details of the TRIM5α antiviral mechanism remain to be solved, but the simplest model is SPRY domain-dependent recognition of incoming capsids and interference with subsequent core maturation and uncoating. It is intriguing that Fv1 and TRIM5α, two proteins with no apparent homology, have antiviral activities against MLV-N, both with specificity for the MLV CA at position 110. It is also striking that despite similar antiviral specificity, Fv1-restricted MLV is able to synthesize DNA, whereas TRIM5α-restricted MLV does not. Here we demonstrate that Fv1 and TRIM5α operate independently and that they can compete for retroviral particles at a pre-reverse transcription step.

Primate lentiviruses have narrow host ranges, due in part to their sensitivities to mammalian intracellular antiviral factors such as APOBEC3G and TRIM5α. Despite the protection provided by this innate immune system, retroviruses are able to transfer between species where they can cause disease. This is true for sooty mangabey simian immunodeficiency virus, which has transferred to humans as HIV-2 and to rhesus macaques as SIVmac, where it causes AIDS. Here we examine the sensitivities of the closely related HIV-2 and SIVmac to restriction by TRIM5α. We show that rhesus TRIM5α can restrict HIV-2 but not the closely related SIVmac. SIVmac has not completely escaped TRIM5α, as shown by its sensitivity to distantly related TRIM5α from the New World squirrel monkey. Squirrel monkey TRIM5α blocks SIVmac infection after DNA synthesis and is not saturable with restriction-sensitive virus-like particles. We map the determinant for TRIM5α sensitivity to the structure in the capsid protein that recruits CypA into HIV-1 virions. We also make an SIV, mutated at this site, which bypasses restriction in all cells tested.

The Fv1 gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the Fv1 gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.

Tripartite motif (TRIM) protein superfamily members are emerging as important effectors of the innate immune response against viral infections. In particular, TRIM22 was reported to exert antiviral activity against RNA viruses, such as hepatitis B virus (HBV), encephalomyocarditis virus (ECMV), and human immunodeficiency virus type 1 (HIV-1). We demonstrate here, for the first time, that TRIM22 is upregulated by influenza A virus (IAV) infection at both mRNA and protein levels in human alveolar epithelial A549 cells. Conversely, TRIM22 potently restricted IAV replication, in that prevention of TRIM22 expression by means of short hairpin RNA led to a 10-fold enhancement of IAV replication in these cells. Depletion of TRIM22 also reduced the anti-IAV activity of alpha interferon (IFN-α), suggesting that TRIM22 is an important IFN-stimulated gene that is required for maximal suppression of IAV by type I IFN. Furthermore, the IAV infectious titer decreased up to 100-fold in MDCK cells expressing exogenous human TRIM22. Restriction of IAV replication was accounted for by the interaction between TRIM22 and the viral nucleoprotein (NP), resulting in its polyubiquitination and degradation in a proteasome-dependent manner. Thus, TRIM22 represents a novel restriction factor upregulated upon IAV infection that curtails its replicative capacity in epithelial cells.

Restriction of murine leukemia virus (MLV) was examined in cells from a range of mammals. All nonmurine restrictions were saturable blocks to N-tropic MLV infection, and several were prior to reverse transcription. We demonstrate restriction in cells from bat and show that if we express the murine restriction factor Fv1 in human cells, then Fv1, not the human host, defines the stage at which infection is blocked.

We have previously described postentry restriction of murine leukemia virus in mammals. Here we characterize the block in human cells. Restricted infection kinetics are multiple hit at high virus dose, and restriction is abrogated by preexposure to restricted virus. We hypothesize that restricted capsid can titrate out the restriction factor.