The human APOBEC3 family of cytidine deaminases constitute an intrinsic defense mechanism that is effective against a range of viruses and retroelements. While these enzymes are well established as antiviral molecules that work independent of cofactors, the possibility of their protein-protein interaction network altering their antiviral activity has not been previously investigated. We analyzed an existing APOBEC3 affinity purification and mass spectrometry (AP-MS) data set and determined that APOBEC3G (A3G) interacts with ZAP, a zinc-finger antiviral protein (also called ZCCHV). ZAP is a Pattern Recognition Receptor (PRR) that binds single-stranded RNA and inhibits viral replication through multiple mechanisms, including targeting RNA for degradation and inhibiting its translation by binding to CpG dinucleotides. Both, A3G and ZAP are known to interact with HIV-1 RNA. ZAP action decreases HIV-1 titres and A3G induces mutations in newly formed HIV-1 (-)DNA. Despite these factors when expressed alone working at different HIV-1 lifecycle stages, we observed synergy between co-expressed A3G and ZAP that resulted in greatly reduced HIV-1 infectious titres from producer cells when compared to ZAP alone. The data suggests that the A3G-ZAP protein-protein interaction is enabling co-restriction in a manner unique from when each protein acts alone. Mechanistic details of this co-restriction will be discussed.

APOBEC3, deoxycytidine/cytidine deaminases, are a seven-member family in humans named APOBEC3A through APOBEC3H, excluding APOBEC3E. APOBEC3s are host restriction factors against retroviruses such as HIV-1. They inhibit replication by deaminating cytosine to uracil in single-stranded DNA during reverse transcription in newly infected CD4+ T cells. The HIV-1 Vif protein interacts directly with APOBEC3 enzymes and induces their ubiquitination and proteasomal degradation to enable viral replication. The Vif-APOBEC3F interaction domain was originally mapped in the C-terminal domain of APOBEC3F. However, in 2021, Nchioua et. al. demonstrated that an N-terminal domain APOBEC3F mutant (R128T) compared to wild-type APOBEC3F exhibited greater Vif resistance and antiviral activity during infection with the HIV-1 Transmitted/Founder virus CH077. This unexpected result suggested that Vif interacted with the N-terminal domain of APOBEC3F. We hypothesized that the N-terminal domain of APOBEC3F had a role in Vif-mediated degradation. We confirmed this through co-immunoprecipitation with APOBEC3F wild type and mutants. In addition, the greater Vif resistance and antiviral activity of APOBEC3F R128T was observed with multiple Transmitted/Founder viruses. Altogether, our study supports a novel Vif interaction interface in the APOBEC3F N-terminal domain and provides new insights into the molecular mechanisms underlying APOBEC3F antiviral activity.

During transmission, a single human immunodeficiency virus 1 (HIV-1) clone, termed the transmitted/founder (T/F) virus, establishes systemic infection within the new host. Our group has previously identified key traits of T/F HIV-1 that contribute to its successful transmission, including variations in envelope glycosylation, and demonstrated that transmission modes—such as those from men who have sex with men (MSM), heterosexual (HET) individuals, and people who inject drugs (PWID)—significantly influence T/F transmission fitness in cervical tissue. Additionally, in ex vivo experiments, we have shown that lectin saturation of genital tissue explants, which prevents glycan binding, enhanced transmission, further emphasizing the role of envelope glycosylation in HIV-1 transmission efficiency.

To investigate the role of glycosylation in HIV-1 transmission fitness, we performed a lectin microarray analysis to compare the glycosylation profiles of T/F Env from derived from different transmission risk groups. While no differences were observed in glycosylation profiles between T/F viruses from different transmission modes, T/F Env showed distinct features compared to chronic Env, including lower levels of bisecting GlcNAc and higher levels of fucosylated glycans. Fucosylated glycans correlated with phenotypic data previously collected, including improved replication capacity in cervical tissue and faster host cell entry. These findings suggest glycosylation patterns, particularly fucosylated and complex glycans, may play a key role in T/F HIV-1’s ability to evade immunity and establish infection in a new host. Future work will focus on systematically modifying the glycan profiles on T/F Env to assess their impact on transmission in human genital tissue explants, as well as using mass spectrometry to further investigate how specific glycosylation structures influence HIV-1 transmission. Glycosylation of the envelope poses a challenge to vaccine design by shielding critical epitopes on the HIV-1 surface. This project aims to characterize the glycan profiles of HIV-1 to inform and advance vaccine development.

Human Immunodeficiency Virus (HIV) persists as a global threat with over 38 million people infected. While the mechanism of HIV viral genomic RNA (vgRNA) packaging is well-studied, vgRNA’s ability to evade the Nonsense Mediated mRNA Decay (NMD) pathway is poorly understood. NMD functions to degrade transcripts that contain Premature Termination Codons (PTCs). Recent studies have demonstrated that NMD-essential UPF1 robustly and nonspecifically binds RNAs and translating ribosomes physically displace UPF1. Despite the full-length vgRNA containing multiple PTCs, many transcripts evade NMD and are selectively packaged into nascent virions. The Arts laboratory has identified a critical RNA secondary structure element termed the Genomic RNA Packaging Enhancer (GRPE) that overlaps the ribosomal frameshifting site needed for translation of the Gag-Pol polyprotein. It is hypothesized the GRPE modulates frameshifting frequency allowing for downstream UPF1 displacement, NMD evasion, and preferential vgRNA packaging. To confirm this, an HIV backbone was modified with luciferase reporters and an inducible promoter to quantitatively measure frameshifting frequency and RNA decay rates, respectively. Additionally, mutations were made to destabilize the GRPE RNA secondary structure, and their frameshifting frequencies, RNA decay rates, and packaging efficiencies were studied. With these mutants, we observed that ribosomal frameshifting was correlated with both RNA decay rates and packaging of the mutant transcript into nascent virions. Ultimately, these results suggest a model of HIV packaging that is concurrent with the translation status of the vgRNA itself and translation may serve as a mechanism of NMD protection. Understanding the mechanism behind HIV NMD evasion can greatly increase lentiviral gene transduction efficiencies as all currently used lentiviral transduction systems do not harbour a GRPE. Preliminary GRPE incorporation increased gene transduction 4-fold as a direct effect of increased packaging, enhancing the plausibility of lentiviral gene transduction to help in the cure of many genetic diseases.

Despite improvements in HIV-1 therapies drug treatment failure and the inability to target latent viral reservoirs remain major limitations. Infection generally requires HIV recognizing the primary receptor CD4, followed by binding to either of the chemokine coreceptors, CXCR4 or CCR5. This binding mode enables cell entry by HIV gp41 fusion with the cell membrane. Investigators have believed for some time that initial binding to CD4 is required for HIV-1 infection. However, studies have shown that HIV-1 can enter cells lacking CD4 although this remains controversial, and the mechanisms are unclear. This study aims to identify a model CD4-negative cell line that is infectable with HIV-1 to study alternate entry mechanisms and determine whether CD4-negative cells can act as latent viral reservoirs. CD4-negative human osteosarcoma (HOS) cells expressing either coreceptor were infected with an envelope-pseudotyped, luciferase or GFP reporter HIV-1.; JR-FL-envelope for CCR5+ and HXB2-envelope for CXCR4+ cells. These cells were confirmed to be CD4 negative through flow cytometry, mRNA expression & the use of a CD4 blocking antibody (Ibalizumab) during infection assays. We found that HIV-1 can infect HOS cells, as demonstrated by post-infection luciferase and GFP expression that was blocked by the integration inhibitor Raltegravir. Infection in HOS cells is resistant to Ibalizumab but susceptible to coreceptor blockers, suggesting that infection is CD4 independent but coreceptor dependent. These results indicate that in the absence of the CD4 receptor, HIV-1 uses an alternative mechanism for viral entry into HOS cells. Further studies will examine infected GFP-positive cells, including single-cell RNA sequencing, determining mechanisms used for viral entry, and the use of a red/green reporter virus to investigate latency. Deepening our understanding of viral entry into CD4-negative cells may help identify unknown viral reservoirs, improve HIV-1 therapeutics, and contribute to finding an eventual cure.

Background: Elite controllers naturally control HIV replication and have small viral reservoirs. Exceptional elite controllers (EEC) present with higher-level control, smaller and replication incompetent reservoirs, and are the model for functional cure. Further elucidation of associated immunological and virological factors is needed. We present an in-depth case study describing the HIV-specific immune responses and viral reservoir in an EEC.
Methods: Viral reservoirs in peripheral CD4+ T cells were quantified by quantitative viral outgrowth assay (qVOA) and intact proviral DNA assay (IPDA). Proviruses were sequenced with FLIP-seq and sub-genomic PCR of immunodominant regions of interest. Ex vivo IFN-y responses were determined using ELISpot assays and peptides spanning the HIV proteome.
Results: The participant EEC is serologically HIV-antibody positive for 6 years, clinically asymptomatic with an undetectable plasma viral load, normal CD4 count, wild-type CCR5 and the protective HLA-B*27:05 allele. qVOA could not detect viral replication after long term culture of more than 600x106 CD4+ T cells. IPDA showed ~1 intact provirus/106 memory CD4+ T cells. FLIP-seq of 1.2x106 central memory CD4+ T cells recovered one defective provirus. Targeted-PCR recovered three Gag sequences and six Nef sequences. All six Nef sequences had a deletion at Nef 75-79 (PLRPM), affecting residues required for MHC-1 downregulation. This Nef deletion could not be detected in >600 subtype-B clinical isolates. The patient has IFN-y responses across the HIV proteome, including a CD8+ response to B*27 Gag KRWIILGLNK and CD4+ response to Pol STAVKAACWWANVTQ.
Conclusion: Despite the absence of active viral replication, this EEC had detectable ex vivo CD4+ and CD8+ responses to multiple regions. The presence of a distinct Nef mutation associated with loss of MHC-1 downregulation suggests ongoing T cell responses are effective in maintaining the EEC phenotype. Our study highlights the importance of Nef inhibition in allowing the immune response to control HIV.

Metabolites, like butyrate and lactate play a crucial role in maintaining gut mucosal homeostasis by enhancing epithelial barrier function and promoting immune tolerance to commensal microbes. Recent findings from the Kaushic lab demonstrated that lactic acid mitigates inflammatory effects of dysbiotic short-chain fatty acids (SCFAs at mM concentrations) and prevents HIV-1 particles from migrating through the VK2 vaginal epithelium. However, evidence in human vaginal mucosa remains sparse. This study hypothesized that HIV-exposed but seronegative (HESN) women exhibit a distinct vaginal metabolite profile that contributes to low immune activation and robust barriers against infection.

We quantified SCFAs, inflammatory mediators, and hydrogen peroxide (H2O2) levels in cervical-vaginal lavage (CVL) samples from Kenyan HIV-seronegative female sex workers (HESN, n=21) and HIV-susceptible controls (n=22). SCFAs, including phenylethylamine (median 23 vs. 16 μM, p=0.02), butyrate (median 26 vs. 12 μM, p<0.0001), and propionate (median 28 vs. 11 μM, p<0.0001), were elevated but still within eubiotic ranges in HESN samples. Our in vitro study showed that butyrate could reduce HIV-1-elicited response of interferon regulatory factor-1(IRF-1), which is key to transactivation of HIV-1 promoter and inflammatory cytokine. Additionally, metabolites like histamine (median 13 vs. 26 μM, p<0.0001), pyruvate (median 13 vs. 26 μM, p=0.0002), acetyl-ornithine (median 14 vs. 25 μM, p<0.001), and tyramine (median 15 vs. 24 μM, p=0.02) were significantly lower in HESN, correlating with reduced levels of pro-inflammatory chemokines (GRO-α, MIG, MCP-1, TGFα, RANTES, TNFα). Consistent with these findings, basal H2O2 levels were elevated in HESN CVL (median 77 vs. 15 μM, p<0.0001), which may inhibit HIV-1 and dampen immune activation. Taken together, these results suggest that vaginal metabolites play a role in mucosal homeostasis, immune modulation, and susceptibility to acquisition of HIV-1 infection.

Introduction: Doravirine (DOR) was designed to retain activity against HIV harboring common NNRTI resistance-associated mutations (RAMs) including K103N, Y181C and G190A. However, the effectiveness of DOR-containing ART combinations in treating individuals with combinations of NNRTI RAMs remains incompletely understood. We assessed DOR phenotypic susceptibility and plasma viral load outcomes in 8 individuals harboring NNRTI RAM combinations who switched to a DOR-containing regimen.
Methods: For each participant, a representative plasma HIV RNA partial pol sequence was single-genome amplified and used to construct a clonal recombinant NL4-3 stock. Viruses were propagated in an immortalized GFP-reporter cell line in the presence of 0.01-10,000nM NNRTIs with EC50 fold-changes (FC) determined relative to NL4.3. Phenotypic DOR resistance was defined as a FC>3.
Results: All control NL4-3 viruses harboring single mutations (K103N/V106A/Y181C/G190A/F227C/ M230L) exhibited NNRTI resistance consistent with the Stanford HIVdb algorithm (v9.6). In contrast, 3/8 clinical isolates displayed lower DOR phenotypic resistance than predicted: participants 66 and 69, predicted as intermediate and low DOR resistance respectively, had FC<3, suggesting a susceptible phenotype, while participant 24, predicted as high DOR resistance, had a FC=4.1 suggesting moderate DOR resistance. All eight participants maintained pVL <40 copies/mL at their last visit while on DOR, including participant 46 who harbored a Y318F substitution conferring high-level DOR resistance as expected. Notably, after a 4-month ART interruption during which pVL rebounded to >100,000 c/mL, participant 46 re-suppressed to 42 c/mL after restarting the same regimen.
Conclusion: Individuals harboring HIV with various common NNRTI RAM combinations can be successfully treated with DOR.