The timing of HIV-1 infection of cells that persist on therapy is not strongly influenced by replication competency or cellular tropism of the provirus.

People with HIV-1 (PWH) on antiretroviral therapy (ART) can maintain undetectable virus levels, but a small pool of infected cells persists. This pool is largely comprised of defective proviruses that may produce HIV-1 proteins but are incapable of making infectious virus, with only a fraction (~10%) of these cells harboring intact viral genomes, some of which produce infectious virus following ex vivo stimulation (i.e. inducible intact proviruses). A majority of the inducible proviruses that persist on ART are formed near the time of therapy initiation. Here we compared proviral DNA (assessed here as 3' half genomes amplified from total cellular DNA) and inducible replication competent viruses in the pool of infected cells that persists during ART to determine if the original infection of these cells occurred at comparable times prior to therapy initiation. Overall, the average percent of proviruses that formed late (i.e. around the time of ART initiation, 60%) did not differ from the average percent of replication competent inducible viruses that formed late (69%), and this was also true for proviral DNA that was hypermutated (57%). Further, there was no evidence that entry into the long-lived infected cell pool was impeded by the ability to use the CXCR4 coreceptor, nor was the formation of long-lived infected cells enhanced during primary infection, when viral loads are exceptionally high. We observed that infection of cells that transitioned to be long-lived was enhanced among people with a lower nadir CD4+ T cell count. Together these data suggest that the timing of infection of cells that become long-lived is impacted more by biological processes associated with immunodeficiency before ART than the replication competency and/or cellular tropism of the infecting virus or the intactness of the provirus. Further research is needed to determine the mechanistic link between immunodeficiency and the timing of infected cells transitioning to the long-lived pool, particularly whether this is due to differences in infected cell clearance, turnover rates and/or homeostatic proliferation before and after ART.

Prevalence of Drug-Resistant Minority Variants in Untreated HIV-1-Infected Individuals With and Those Without Transmitted Drug Resistance Detected by Sanger Sequencing.

Minority variant human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are associated with an increased risk of virological failure during treatment with NNRTI-containing regimens. To determine whether individuals to whom variants with isolated NNRTI-associated drug resistance were transmitted are at increased risk of virological failure during treatment with a non-NNRTI-containing regimen, we identified minority variant resistance mutations in 33 individuals with isolated NNRTI-associated transmitted drug resistance and 49 matched controls. We found similar proportions of overall and nucleoside reverse transcriptase inhibitor-associated minority variant resistance mutations in both groups, suggesting that isolated NNRTI-associated transmitted drug resistance may not be a risk factor for virological failure during treatment with a non-NNRTI-containing regimen.

Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance.

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues.

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses.

Next generation sequencing (NGS)/deep sequencing has become an important tool in the study of viruses. The use of unique molecular identifiers (UMI) can overcome the limitations of PCR errors and PCR-mediated recombination and reveal the true sampling depth of a viral population being sequenced in an NGS experiment. This approach of enhanced sequence data represents an ideal tool to study both high and low abundance drug resistance mutations and more generally to explore the genetic structure of viral populations. Central to the use of the UMI/Primer ID approach is the creation of a template consensus sequence (TCS) for each genome sequenced. Here we describe a series of experiments to validate several aspects of the Multiplexed Primer ID (MPID) sequencing approach using the MiSeq platform. We have evaluated how multiplexing of cDNA synthesis and amplicons affects the sampling depth of the viral population for each individual cDNA and amplicon to understand the relationship between broader genome coverage versus maximal sequencing depth. We have validated reproducibility of the MPID assay in the detection of minority mutations in viral genomes. We have also examined the determinants that allow sequencing reads of PCR recombinants to contaminate the final TCS data set and show how such contamination can be limited. Finally, we provide several examples where we have applied MPID to analyze features of minority variants and describe limits on their detection in viral populations of HIV-1 and SARS-CoV-2 to demonstrate the generalizable utility of this approach with any RNA virus.

Structural Analysis of Potent Hybrid HIV-1 Protease Inhibitors Containing Bis-tetrahydrofuran in a Pseudosymmetric Dipeptide Isostere.

The design, synthesis, and X-ray structural analysis of hybrid HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran (bis-THF) in a pseudo-C2-symmetric dipeptide isostere are described. A series of PIs were synthesized by incorporating bis-THF of darunavir on either side of the Phe-Phe isostere of lopinavir in combination with hydrophobic amino acids on the opposite P2/P2' position. Structure-activity relationship studies indicated that the bis-THF moiety can be attached at either the P2 or P2' position without significantly affecting potency. However, the group on the opposite P2/P2' position had a dramatic effect on potency depending on the size and shape of the side chain. Cocrystal structures of inhibitors with wild-type HIV-1 protease revealed that the bis-THF moiety retained similar interactions as observed in the darunavir-protease complex regardless of the position on the Phe-Phe isostere. Analyses of cocrystal structures and molecular dynamics simulations provide insights into optimizing HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide isosteres.

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2' Ligands To Optimize Hydrogen Bonding in the Substrate Envelope.

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.

Picomolar to Micromolar: Elucidating the Role of Distal Mutations in HIV-1 Protease in Conferring Drug Resistance.

Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 µM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.

Rationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies.

An array of carbohydrates masks the HIV-1 surface protein Env, contributing to the evasion of humoral immunity. In most HIV-1 isolates 'glycan holes' occur due to natural sequence variation, potentially revealing the underlying protein surface to the immune system. Here we computationally design epitopes that mimic such surface features (carbohydrate-occluded neutralization epitopes or CONE) of Env through 'epitope transplantation', in which the target region is presented on a carrier protein scaffold with preserved structural properties. Scaffolds displaying the four CONEs are examined for structure and immunogenicity. Crystal structures of two designed proteins reflect the computational models and accurately mimic the native conformations of CONEs. The sera from rabbits immunized with several CONE immunogens display Env binding activity. Our method determines essential structural elements for targets of protective antibodies. The ability to design immunogens with high mimicry to viral proteins also makes possible the exploration of new templates for vaccine development.

HIV-1 Protease Uses Bi-Specific S2/S2' Subsites to Optimize Cleavage of Two Classes of Target Sites.

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1' proline. A P1' proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1'-P2'), with two complementary sets of rules. When P1' is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2' amino acid interacts with a hydrophobic region of the S2' subsite. When P1' is not proline, the orientations of the P2 and P2' side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2' amino acid usually engages hydrophilic amino acids in the S2' subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2' subsites to accommodate the steric effects imposed by a P1' proline on the orientation of P2 and P2' substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay.

BACKGROUND: In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA). METHODS: Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid antigen positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1-V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with highly active antiretroviral therapy in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution. RESULTS: IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r = 0.94; P < 0.0001). CONCLUSIONS: VOA-UDSA may be an alternative readout for that currently used for QVOA.