BACKGROUND:Human immunodeficiency virus (HIV) establishes persistent infection by integrating its proviral DNA into the host genome. While most integrated proviruses are defective, a small portion of proviruses are intact that represent a major obstacle to achieving an HIV cure. We have designed an approach for selective elimination of host cells harboring replication-competent HIV (SECH), through inhibition of autophagy and anti-apoptotic molecules during viral reactivation. However, the effects of SECH on the dynamics of intact and defective HIV provirus remain unclear. METHODS:We isolated DNA from HIV-infected samples after SECH treatments. We analyzed HIV type 1 (HIV-1) proviruses in these samples by intact proviral DNA assay, near full-length polymerase chain reaction (PCR), and DNA sequencing analyses. RESULTS:We show that SECH treatments reduce reservoirs harboring intact but not defective HIV proviruses by intact proviral DNA assay. Nested PCR and DNA sequencing analyses confirm that SECH treatments can delete full-length HIV-1 proviruses in humanized mice in vivo and in patient samples ex vivo. CONCLUSIONS:Our data suggest that the SECH method is capable of effectively targeting HIV reservoirs harboring intact HIV proviruses that can restart active viral replication.

Objectives HIV reservoirs in myeloid cells, including tissue-resident macrophages, persist despite antiretroviral therapy, hindering viral eradication. We have tested an approach for HIV clearance through selective elimination of host cells harboring replication-competent HIV (SECH), by inhibition of autophagy and anti-apoptotic molecules during viral reactivation. Whether the SECH approach can effectively target reservoirs of the myeloid lineage is investigated. Methods We examined whether SECH treatments could deplete HIV-infected macrophages from HIV+ donors ex vivo, and in humanized mice in vivo. We also performed spatial transcriptome analyses of myeloid HIV reservoirs in the brains of humanized mice that escaped SECH treatments. Results SECH treatments can eliminate HIV-infected macrophages from HIV+ donors ex vivo, and in a portion of humanized mice in vivo. Spatial transcriptome analyses showed increases in epigenetic repressors for HIV transcription, as well as elevated autophagy and glycolysis genes in brain myeloid cells resistant to SECH treatments, while counteracting these molecular mechanisms sensitized the myeloid reservoirs to cell death. Conclusions Our data suggest that myeloid HIV reservoirs resistant to depletion display limited HIV gene expression by epigenetic repressors, and express more pro-survival genes, while targeting these mechanisms helps to promote the clearance of myeloid HIV reservoirs.

Drug resistance remains a significant obstacle to identifying effective treatments for HIV-1 infection. Integrase strand transfer inhibitors (INSTIs) are frontline treatments used in combination antiretroviral therapy, but their efficacy can be compromised by emergence of resistance-associated mutations. The development of compounds that will retain efficacy against drug-resistant variants is of significant interest. Herein, we report the synthesis of naphthyridine-based INSTIs with a combination of 4-amino and 5-hydroxymethyl groups. Comparison of the resistance mutant profiles of the new compounds with FDA-approved second-generation INSTIs showed that the lead compounds are comparable to or surpass the efficacy of clinically used drugs against some of the most prevalent drug-resistant mutations that emerge in patient-derived viral isolates. High-resolution cryogenic electron microscopy (cryo-EM) structures of HIV-1 intasomes with two of the best naphthyridine-based INSTIs bound highlight how these inhibitors make enhanced interactions with viral DNA, particularly through optimized DNA stacking. Molecular dynamics simulations together with quantum mechanical and molecular mechanical calculations indicate that the interplay between intramolecular bonding, stacking geometry, resonance effects, and charge distribution governs drug binding within the active site of the intasome. The data mechanistically explain how key interactions contribute to improved antiviral potency against drug-resistant mutants and highlight a new strategy to combat HIV-1 resistance.

IntroductionHIV integrates its proviral DNA into the host genome to establish persistent infection. To promote HIV clearance, we have designed an approach for selective elimination of host cells harboring replication-competent HIV (SECH), through inhibition of autophagy and anti-apoptotic molecules during viral reactivation. SECH approach can clear HIV-infected cells in approximately 50% humanized mice. However, the mechanisms for the resistance of reservoirs to depletion in mice with failure in HIV clearance are unclear.MethodsWe have performed single cell transcriptome analyses of HIV-infected T cells that escaped the treatments, in order to identify cellular pathways that could be targeted to facilitate the deletion of refractory HIV reservoirs.ResultsBy single cell RNA sequencing analyses of T cell reservoirs resistant to SECH treatments, we found increases in pro-survival autophagy and glycolysis. Moreover, these resistant reservoirs expressed more epigenetic modifiers that repress HIV gene expression, while targeting such epigenetic repression promoted cell death in HIV-infected cells.DiscussionOur results indicate that T cell reservoirs refractory to depletion maintain a delicate balance between low levels of HIV gene expression and evasion of cell death. This study suggests that targeting epigenetic repression of HIV is critical for the depletion of the viral reservoirs.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the IN tetramer is an important antiviral target for investigational allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions and suggest a working model for IN-viral RNA binding. Moreover, our structure of the IN tetramer provides atomic blueprints for the rational development of improved allosteric inhibitors.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271–276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

HIV-1 integrase (IN) is an important molecular target for the development of anti-AIDS drugs. A recently FDA-approved second-generation integrase strand transfer inhibitor (INSTI) cabotegravir (CAB, 2021) is being marketed for use in long-duration antiviral formulations. However, missed doses during extended therapy can potentially result in persistent low levels of CAB that could select for resistant mutant forms of IN, leading to virological failure. We report a series of N-substituted bicyclic carbamoyl pyridones (BiCAPs) that are simplified analogs of CAB. Several of these potently inhibit wild-type HIV-1 in single-round infection assays in cultured cells and retain high inhibitory potencies against a panel of viral constructs carrying resistant mutant forms of IN. Our lead compound, 7c, proved to be more potent than CAB against the therapeutically important resistant double mutants E138K/Q148K (>12-fold relative to CAB) and G140S/Q148R (>36-fold relative to CAB). A significant number of the BiCAPs also potently inhibit the drug-resistant IN mutant R263K, which has proven to be problematic for the FDA-approved second-generation INSTIs.

Vibrio vulnificus, a significant marine pathogen, undergoes opaque (Op)-translucent (Tr) colony switching based on whether capsular polysaccharide (CPS) is produced. CPS phase variation is sometime accompanied by genetic variation or down-regulation of particular genes, such as wzb. In addition, CPS prevents biofilm formation and is important to the virulence of V. vulnificus. However, the extent to which there is a difference in gene expression between Tr and Op colonies and the impact of CPS phase variation on other behaviors of V. vulnificus remain unknown. In this work, the data have shown that CPS phase variation of V. vulnificus is affected by incubation time. Tr and Op strains exhibited similar growth rates. However, Tr strains had enhanced biofilm formation capacities but reduced swimming motility compared to Op strains. The RNA-seq assay revealed 488 differentially expressed genes, with 214 downregulated and 274 upregulated genes, between Tr and Op colonies. Genes associated with Tad pili and CPS were downregulated, whereas those involved in flagellum were upregulated, in Tr colonies compared with Op colonies. In addition, 9 putative c-di-GMP metabolism-associated genes and 28 genes encoding putative regulators were significantly differentially expressed, suggesting that CPS phase variation is probably strictly regulated in V. vulnificus. Moreover, 8 genes encoding putative porins were also differentially expressed between the two phenotypic colonies, indicating that bacterial outer membrane was remodeled during CPS phase variation. In brief, this work highlighted the gene expression profiles associated with CPS phase variation, but more studies should be performed to disclose the intrinsic mechanisms in the future.

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

The L-arabinose inducible pBAD vectors are commonly used to turn on and off the expression of specific genes in bacteria. The utilization of certain carbohydrates can influence bacterial growth, virulence factor production, and biofilm formation. Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, can grow in media with L-arabinose as the sole carbon source. However, the effects of L-arabinose on V. parahaemolyticus physiology have not been investigated. In this study, we show that the growth rate, biofilm formation capacity, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus are negatively affected by L-arabinose. RNA-seq data revealed significant changes in the expression levels of 752 genes, accounting for approximately 15.6% of V. parahaemolyticus genes in the presence of L-arabinose. The affected genes included those associated with L-arabinose utilization, major virulence genes, known key biofilm-related genes, and numerous regulatory genes. In the majority of type III secretion system, two genes were upregulated in the presence of L-arabinose, whereas in those of type VI secretion system, two genes were downregulated. Ten putative c-di-GMP metabolism-associated genes were also significantly differentially expressed, which may account for the reduced c-di-GMP levels in the presence of L-arabinose. Most importantly, almost 40 putative regulators were significantly differentially expressed due to the induction by L-arabinose, indicating that the utilization of L-arabinose is strictly regulated by regulatory networks in V. parahaemolyticus. The findings increase the understanding of how L-arabinose affects the physiology of V. parahaemolyticus. Researchers should use caution when considering the use of L-arabinose inducible pBAD vectors in V. parahaemolyticus. IMPORTANCE The data in this study show that L-arabinose negatively affects the growth rate, biofilm formation, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus. The data also clarify the gene expression profiles of the bacterium in the presence of L-arabinose. Significantly differentially expressed genes in response to L-arabinose were involved in multiple cellular pathways, including L-arabinose utilization, virulence factor production, biofilm formation, motility, adaptation, and regulation. The collective findings indicate the significant impact of L-arabinose on the physiology of V. parahaemolyticus. There may be similar effects on other species of bacteria. Necessary controls should be established when pBAD vectors must be used for ectopic gene expression.

The HIV-1 reservoirs harbor the latent proviruses that are integrated into the host genome. It is a challenging task to eradicate the proviruses in order to achieve an HIV cure. We have described a strategy for the clearance of HIV-1 infection through selective elimination of host cells harboring replication-competent HIV (SECH), by inhibition of autophagy and promotion of apoptosis during viral re-activation. HIV-1 can infect various CD4+ T cell subsets, but it is not known whether the SECH approach is equally effective in targeting HIV-1 reservoirs in these different subsets in vivo. In a humanized mouse model, we found that treatments of HIV-1 infection by suppressive antiretroviral therapy (ART) led to the establishment of latent HIV reservoirs in naïve, central memory and effector memory T cells. Moreover, SECH treatments could clear latent HIV-1 reservoirs in these different T cell subsets of humanized mice. Co-culture studies showed that T cell subsets latently infected by HIV-1, but not uninfected bystander cells, were susceptible to cell death induced by SECH treatments. Our study suggests that the SECH strategy is effective for specific targeting of latent HIV-1 reservoirs in different T cell subsets.

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Mitochondrial disruption leads to the release of cytochrome c to activate caspase-9 and the downstream caspase cascade for the execution of apoptosis. However, cell death can proceed efficiently in the absence of caspase-9 following mitochondrial disruption, suggesting the existence of caspase-9-independent cell death mechanisms. Through a genome-wide siRNA library screening, we identified a network of genes that mediate caspase-9-independent cell death, through ROS production and Alox5-dependent membrane lipid peroxidation. Erk1-dependent phosphorylation of Alox5 is critical for targeting Alox5 to the nuclear membrane to mediate lipid peroxidation, resulting in nuclear translocation of cytolytic molecules to induce DNA damage and cell death. Consistently, double knockouts of caspase-9 and Alox5 in mice, but not deletion of either gene alone, led to significant T cell expansion with inhibited cell death, indicating that caspase-9- and Alox5-dependent pathways function in parallel to regulate T cell death in vivo. This unbiased whole-genome screening reveals an Erk1-Alox5-mediated pathway that promotes membrane lipid peroxidation and nuclear translocation of cytolytic molecules, leading to the execution of cell death in parallel to the caspase-9 signaling cascade.

The formation of long-lived immune memory cells specific for pathogens is critical for the establishment of long-term immune protection against future infections. BNIP3L/NIX and BNIP3, two functionally redundant BCL2 family members required for mitophagy, undergo significant upregulation after memory B cells are formed. Deletion of Bnip3l and Bnip3 leads to mitochondrial accumulation, and increases in oxidative phosphorylation and fatty acid synthesis, resulting in the loss of memory B cells. These observations suggest that after the formation of memory B cells, mitophagy is critical for clearing superfluous mitochondria to re-shape the metabolic programs, thereby protecting the metabolic quiescence and longevity of memory B cells .

The development of long-lived immune memory cells against pathogens is critical for the success of vaccines to establish protection against future infections. However, the mechanisms governing the long-term survival of immune memory cells remain to be elucidated. In this article, we show that the maintenance mitochondrial homeostasis by autophagy is critical for restricting metabolic functions to protect IgG memory B cell survival. Knockout of mitochondrial autophagy genes, Nix and Bnip3, leads to mitochondrial accumulation and increases in oxidative phosphorylation and fatty acid synthesis, resulting in the loss of IgG+ memory B cells in mice. Inhibiting fatty acid synthesis or silencing necroptosis gene Ripk3 rescued Nix-/-Bnip3-/- IgG memory B cells, indicating that mitochondrial autophagy is important for limiting metabolic functions to prevent cell death. Our results suggest a critical role for mitochondrial autophagy in the maintenance of immunological memory by protecting the metabolic quiescence and longevity of memory B cells.