Background: In cisgender females (cF), the vaginal microbiota play a critical role in STI and HIV acquisition. An optimal cF vaginal microbiota is dominated by Lactobacillus spp., while microbiota dominated by diverse anaerobic bacteria are associated with inflammation and molecular bacterial vaginosis (BV). Local environmental factors shape the microbiome. This is demonstrated by the effect of circumcision on the microbiota composition of the coronal sulcus in cisgender men (cM) which increases water loss and oxygen tension, decreasing the abundance of strict anaerobes. Penile inversion vaginoplasty is the primary surgical technique used to create a neovagina however, the influence of surgical invagination, of what was penile tissue, on the microbiome of the transfeminine (tF) neovagina is poorly understood.

Methods: Self-collected neovaginal swabs from n=60 tF individuals who underwent vaginoplasty >1 year prior were analyzed. Microbiota were characterized through amplification, sequencing, and bioinformatic analyses of the V3-V4 region of the 16S rRNA gene. tF neovaginal microbiota profiles were compared to those of the reproductive aged cF vagina (n=100) and the sub-preputial space of uncircumcised cM (n=100).

Results: The microbiota of the tF neovagina did not resemble the Lactobacillus dominated or molecular BV-like cF vagina, but instead had high abundance of Gram-negative anaerobes (e.g., Prevotella, Porphyromonas, Anaerococcus, and Dialister).

Conclusions: Our early findings suggest the tF neovaginal microbiome has a low abundance of Lactobacillus spp. and instead is dominated by species like those found in the coronal sulcus of uncircumcised cM. Given the importance of the vaginal microbiota in HIV susceptibility of cF, future work will investigate relationships between microbiota, inflammation, and behavioural practices.

A contributing factor to HIV-1 persistence is its ability to evade host innate and adaptive immunity. The host restriction factor SERINC5 restricts HIV-1 virion infectivity by incorporating into the viral membrane during egress. To overcome this, HIV-1 Nef interacts with Adaptor Protein 2 (AP-2), which triggers cell surface SERINC5 internalization, thereby preventing virion incorporation and restoring infectivity. While it is understood that internalized SERINC5 is ultimately re-routed to the lysosome where it is degraded, the specific trafficking pathways HIV-1 Nef hijacks to direct internalized SERINC5 to the lysosome remains unclear. Interestingly, Nef utilizes its [D/E]xxxLL₁₆₇ dileucine motif to interact with a variety of Adaptor Proteins (APs) at various locations within the endolysosomal network to facilitate specific trafficking events in a temporal and spatial manner.

Herein, we describe an ND₁₆₄ Nef polymorphism occurring within the HIV-1 Nef dileucine motif that uncouples cell surface SERINC5 downregulation from a cognate Nef function, CD4 downregulation. The presence of the ND₁₆₄ polymorphism results in a significant decrease in infectious virus yield in the presence of SERINC5 compared to Nef isolates lacking this polymorphism, suggesting this region of Nef is key for SERINC5-dependent restriction. To decipher the mechanism(s) behind this restriction, we will first define the molecular interface between Nef and the various APs present within the endolysosomal system. Subsequent siRNA-based experiments will then be conducted to determine which APs are likely involved in Nef-mediated re-routing of internalized SERINC5 to the lysosome.

Overall, these studies will define the precise trafficking itinerary commandeered by Nef to downregulate cell surface SERINC5. Characterizing these trafficking pathways Nef hijacks to downregulate SERINC5 could reveal an ‘Achilles Heel’ that could be exploited using novel therapeutics designed to block such interactions.

Nef is an intrinsically disordered accessory protein unique to the primate lentiviruses (PLV), including HIV-1, HIV-2 and SIV. Nef has several interactions with host cellular proteins to enhance virus replication, including antagonistic binding of tetherin and downregulation of CD4, MHC-I and the SERINC5 restriction factor. In this study, we reconstructed the nef sequence of the PLV ancestor and characterized its function in cellulo.

We used BEAST to sample time-scaled phylogenies relating 34 PLV Nef amino acid sequences from the posterior distribution incorporating prior information on the origin of PLVs. We sampled 1,000 trees from the converged chain samples and then reconstructed ancestral sequences using Historian, which models both substitution and indel events. Next, we extracted the best supported evolutionary pathway from the root to HIV-1 group M and then synthesized the corresponding nucleotide sequences (GeneArt Gene Synthesis, ThermoFisher). Sequences were amplified and cloned into the pN1 expression vector such that expressed Nef is fused to eGFP fluorophore. SERINC5 was exogenously expressed in trans using an HA-tagged vector. CD4+ HeLa cells were plated 24h and then co-transfected with Nef-eGFP and SERINC5 plasmids using PolyJet. After 24h, cells were processed for cell surface staining with conjugated antibodies for flow cytometry analysis, and compared to HIV-1 and SIVmac259 controls.

Reconstructed Nef sequences tended to become shorter along lineages leading to HIV-1/M with increasing disorder. At the root, the mean length was 225 (IQR 206-231) amino acids. The dileucine motif was conserved across all ancestral lineages leading to HIV-1/M. Downregulation of SERINC5 by the synthetic PLV ancestral Nef was not significantly different from SIVmac259 or HIV-1 (P>0.05), but downregulation of surface CD4 was significantly reduced (P<10–4).

Our findings suggest that Nef-mediated CD4 downregulation and intrinsic disorder were likely essential for the establishment of HIV-1/M in humans.

The HIV-1 accessory protein, Nef, plays a central role in HIV virulence and eventual progression to AIDS. Nef contributes to HIV pathogenesis by impairing T cell activation and maturation, subverting apoptosis, and down-regulating cell surface major histocompatibility complex class I (MHC-I) expression. Through its interaction with phosphofurin acidic cluster sorting protein-2 and localization to the trans-Golgi network (TGN), Nef interacts with Src family tyrosine kinases (SFK) to initiate a misdirected signaling cascade that culminates in MHC-I internalization and TGN sequestration. Collectively, Nef-mediated MHC-I down-regulation minimizes immune recognition of HIV-1-infected cells and is a key factor for why a functional cure for HIV remains elusive. We identified a small molecule, H3-1, that was predicted through structure-function design to disrupt the Nef:SFK interaction and exploit this interaction as a target for therapeutic intervention. H3-1 counteracted Nef-dependent MHC-I down-regulation, resulting in measurable improvements in cell surface MHC-I expression in HIV-1-infected primary human and mouse CD4+ T cells in the absence of cytotoxicity. Therefore, we assessed the feasibility of H3-1 treatment to enhance antigen presentation in a transgenic mouse model of AIDS-like disease characterized by Nef expression in CD4+ T cells. In preliminary studies, H3-1 was rapidly cleared in vivo as assessed by mass spectrometry; however, ex vivo cultured transgenic mouse-derived CD4+ splenocytes exhibited enhanced presentation of cell surface MHC-I when complexed to a model epitope following H3-1 treatment. Alongside organic synthesis to generate H3-1 analogues with improved in vivo pharmacokinetics, future studies will evaluate the adjuvant role of H3-1-mediated enhanced antigen presentation in the context of a dendritic cell-based vaccine strategy.

Background: Understanding the longevity of proviruses persisting during ART is critical to HIV eradication, but few such studies have discriminated between intact and defective proviruses. We in-depth characterize the genetic features and inferred ages of proviruses persisting during long-term ART in two individuals.

Methods: Longitudinal pre-ART plasma HIV RNA nef sequences were collected by single-template sequencing from participants P1 and P2, over 4.75 and 2.5 years, respectively. Single-template near-full-length proviral sequences were collected at a single time point ≥9 years after ART initiation and classified as intact or defective. Maximum-likelihood phylogenies were constructed from pre-ART and intact proviral nef sequences, where the ages of the latter sequences were inferred using a root-to-tip regression approach.

Results: A total of 122 and 221 pre-ART nef sequences, and 733 and 386 near-full-length proviruses were recovered from P1 and P2, respectively. Defective proviruses dominated in both individuals (98%). Heavily-deleted proviruses made up most of P2’s proviral pool (90%), while P1’s was extensively hypermutated (53%). In both individuals, gag (P1-27%, P2-38%) and nef (P1-16%, P2-24%) were most likely to be intact. Clonal proviruses made up 17% (P1) and 36% (P2) of sampled sequences. Age inference was performed on proviruses with intact nef. Of these, 97% (P1) and 78% (P2) were estimated to have integrated in the two years pre-ART, consistent with skewing of the proviral pool towards younger sequences. Nevertheless, proviruses dating to the first year of infection were recovered in both participants. No difference was observed in the inferred age distributions of intact and defective proviruses in either participant (p>0.4).

Conclusion: Genetic features of proviruses persisting on ART can vary widely between individuals. The lack of difference in the ages of intact and defective proviruses suggests that genetic integrity is not the most critical feature that modulates proviral longevity after many years on ART.

Background: Liquid-liquid phase separation (LLPS) by multivalent interactions between proteins and nucleic acids generates membraneless, biomolecular condensates (BMCs) compartmentalizing and concentrating specialized molecules that nucleate and drive fundamental biological processes. While emerging evidence indicates that many viruses rely on LLPS for their replication, virus-engineered BMCs remain to be functionally characterized. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) protein phase separates to assemble as BMCs favoring replication by regulating genomic RNA positioning and trafficking. Hypothesis: With retroviral proteins representing those having the highest degree of disorder promoting their phase separation, we hypothesized that the core viral protein components of mature virions may co-condense to themselves become BMCs that promote virus ingress and infectivity. Methods: Using rigorous combinations of biochemical assays and quantitative live-cell imaging techniques, we explore the predisposition, mechanisms, dispersion, and pharmacologic sensitivity of human immunodeficiency virus-type 1 (HIV-1) BMCs. Results: Fluorescence recovery after photobleaching of HIV-1 core proteins is proportional to their degrees of disorder. The mostly disordered HIV-1 NC protein represents a scaffolding condensate onto which HIV-1 capsid, reverse transcriptase and integrase accumulate as client condensates to form mobile BMCs that are trafficked along cytoskeletal networks and accumulate in nuclei. These HIV-1 BMCs are also sensitive to treatment with NC zinc-ejecting drug, azodicarbonamide. We demonstrate that while full-length Gag does not readily phase separate in cells; addition of HIV-1 protease inducing its maturation leads to condensation of proteolytic Gag products. Finally, we discover that intrinsically disordered viral core proteins phase separate and self-assemble with the viral genomic RNA in vitro to attain positioning and geometry characteristic of a viral reverse transcription complex. Conclusion and Perspectives: The finding that HIV-1 core proteins co-condense to attain virus-like architecture redefines the virion cores as BMCs. This study responds to the critical challenge of advancing anti-viral therapies targeting viral BMCs.

Since existing drugs target viral enzymes and entry proteins, innovative means of enhancing existing therapeutics requires identification of host factors or processes critical for the virus. Given HIV-1’s reliance on host alternative splicing for replication, modulators of this process could serve as novel therapeutics to complement and/or enhance existing drugs. Screening of several kinase inhibitors that affect key splicing regulatory factors (SR proteins) identified a β-carboline alkaloid compound, Harmine, that suppressed HIV-1 at low micromolar concentrations. Harmine reduced HIV-1 unspliced and singly-spliced RNAs abundance with limited impact on multiply-spliced RNAs. Treatment of primary CD4+T cells from healthy donors infected with HIV-1 resulted in ~50% reduction in viral structural proteins and altered accumulation of viral RNAs. Although Harmine is a known inhibitor of DYRK1A and monoaminoxidase A, subsequent studies determined that the antiviral effect was independent of effects on either enzyme. Addition of Harmine to primary CD4+T cells selectively altered the expression of host SR proteins and several SR kinases, increasing CLK1 while reducing CLK2 kinase levels. Parallel studies involving depletion of CLK1 or CLK2 determined that loss of CLK1 increases HIV-1 expression while CLK2 depletion reduces it, suggesting that Harmine’s anti-HIV-1 activity could be mediated through changes in the relative activity of these CLKs. To test pan-antiviral activity of Harmine, we assessed its activity against multiple coronaviruses, including SARS-Cov2. Harmine treatment one h post infection (hpi) suppressed replication of both Human-229E and SARS-Cov2 coronaviruses as demonstrated by reduced viral nucleocapsid protein expression and genomic RNA release in media. Delaying compound addition by 16 hpi yielded a similar inhibition of virus replication, suggesting that harmine affects post-entry processes. In addition, cells pre-treated with Harmine had reduced capacity to support coronavirus replication. Together, our data establishes the feasibility of manipulating cellular processes to control the replication of multiple human pathogens of concern.

The APOBEC3 family of cytidine deaminases includes five members that have been reported to inhibit HIV-1 in absence of its Vif protein. HIV-1 Vif antagonizes APOBEC3 proteins by inducing their ubiquitination and degradation through proteasome. Previous studies have identified several domains of Vif that interact with APOBEC3 proteins. However, there is a lack of studies on the effect of Vif on co-expressed APOBEC3 proteins, such as would occur in HIV infected cells. One study from our lab has shown that APOBEC3G and APOBEC3F have the ability to interact and form a hetero-oligomer. This APOBEC3F/APOBEC3G (A3F/A3G) hetero-oligomer was found to be more resistant to Vif mediated degradation from the HIV-1 LAI molecular clone that originated from an HIV-1 positive patient at an unknown time after transmission. However, HIV-1 transmitted/founder (T/F) virus molecular clones that were isolated from patient samples within 6 weeks of infection are now available. Analysis of amino acid sequences of Vif protein of these TF viruses revealed several differences from HIV-1 LAI. Analysis of multiple TF viruses using an APOBEC3 degradation assay revealed variability in Vif mediated degradation of individual APOBEC3 enzymes. Moreover, the A3F/A3G hetero-oligomer showed variability in sensitivity to Vif from different TF viruses. Our results also show that when bound to APOBEC3G, APOBEC3F is partially protected from TF virus Vif mediated degradation. The A3F/A3G hetero-oligomer still interacts with Vif, thus the mechanistic basis of partial resistance appears to be linked to ubiquitination. A better understanding of the APOBEC3/Vif interface and exploration of the mechanisms resulting in the variability of APOBEC3 degradation induced by TF viruses will help in determining the contribution and consequences of APOBEC3 activity during a clinical HIV infection.