Multiple genome-wide knockout/knockdown studies of viral infection have identified sets of host dependency factors (HDFs) that are essential for viral replication. Although these factors may be candidates for developing novel antivirals, defining candidates that do not lead to drug toxicity is challenging. One opportunity to identify promising targets is to leverage available human genome resources to determine which HDFs harbour homozygous loss of function polymorphisms in healthy people. To that end, we sought to combine data from 27 genome-wide host dependency factor screens that covered HIV, Hepatitis C, Hepatitis D, SARS-CoV-2, SARS-CoV, Ebola, Influenza A, Zika, Dengue and West Nile virus, with the genome aggregation database (gnomAD) including >125,000 human exome and >15,000 whole-genome sequences. We identified 2,907 unique HDFs combined across all viruses, including 353 which were essential for ≥2 viruses and 2 which were essential for 5 viruses. Of the combined list, 137 targets were deemed non-essential by the observed/expected constraint score and the presence of homozygous loss-of-function variants found within the gnomAD control population. When functionally annotated, the 2,907 unique HDFs were enriched for several biological processes, notably protein transport, macromolecule catabolism, autophagy, and vacuole organisation. We also found that HDFs implicated in more than one virus were highly intolerant to a loss of function variant, suggesting they are likely to be involved in host-essential processes. In silico and in vitro screening of HDFs harbouring homozygous loss of function variants in healthy people may aid in developing novel broad-acting antivirals capable of targeting more than one virus.

A recent genome-wide association study (GWAS) in 3886 individuals of African ancestry identified genetic variants close to the gene CHD1L to be strongly associated with decreased HIV set-point viral load (spVL) and that CHD1L knockout enhanced HIV replication in myeloid cell models. However, the precise genetic variants that mediate the observed effect of CHD1L, and the extent of cell-specificity remains unclear. To address this, we performed a functionally-informed fine-mapping study of the CHD1L region. Using multiple gene expression datasets from individuals of African ancestry, we observed no significant colocalization of GWAS signals and variants regulating CHD1L expression (eQTLs) in LCLs (N=1,032), whole blood (N=757), or monocytes (N=233) suggesting that the individual effects of variants identified in the GWAS on CHD1L expression may be small. Statistical fine-mapping of the CHD1L region demonstrated that combinations of GWAS variants, rs7519713/rs73004025 and rs7519713/rs72999634, were more strongly associated with HIV spVL than the top GWAS hit alone (ANOVA p=0.023 and p=0.019, respectively) highlighting the enhanced effect of variant combinations on restricting HIV spVL. We then tested how variant combinations may impact CHD1L expression but observed no significant CHD1L expression differences in LCLs (N=89) or monocytes (N=118) in individuals carrying the alternate allele, however small sample sizes limit our power to detect significant differences. We next generated genetically regulated expression data in the 3886 African individuals from the spVL GWAS using two separate training datasets from whole blood or monocytes from African American individuals. Combinations of rs7519713/rs73004025 or rs7519713/rs72999634 were significantly associated with increased CHD1L expression in monocytes (both p<2e-16) but were associated with decreased CHD1L expression in whole blood (p<2e-16). These results suggest that CHD1L-mediated HIV restriction likely occurs in a cell-type specific manner supporting the hypothesis that CHD1L is a cell-specific HIV restriction factor mainly active in the monocyte compartment.

Elucidating the transmitted/founder (T/F) HIV sequence can help us address key questions in HIV evolutionary dynamics at the between-host level (e.g. the nature of the transmission bottleneck) and the within-host level (e.g. the ancestor-descendant relationships between the T/F virus and HIV sequences persisting in the reservoir). T/F sequences should ideally be reconstructed from plasma HIV RNA sequences collected immediately after seroconversion (which is not feasible in most settings) or from plasma HIV RNA sequences sampled longitudinally during untreated infection. The latter approach however is longer possible because current guidelines recommend that antiretroviral treatment (ART) be initiated immediately following HIV diagnosis. Alternative methods to infer T/F virus sequences are therefore needed. Reasoning that the population of proviruses that persist in an individual during long-term ART represent an archival record of within-host HIV evolution, we explored the possibility that these sequences could be used to phylogenetically infer the T/F sequence (or a close descendant thereof). To do this, we leveraged HIV sequence data sets from 11 individuals that comprised single-genome plasma HIV RNA env sequences collected shortly after seroconversion (which served as a proxy for the T/F virus sequence) along with single-genome proviral sequences collected during suppressive ART (median 23; interquartile range 10-25.5 unique proviruses/participant). We explored a variety of phylogenetic methods to estimate ancestral sequences, including phangorn, IQ-TREE and MrBayes, to investigate whether proviral sequences can be used to recover T/F viruses sequences. All methods produced broadly comparable results. Notably, we were able to correctly reconstruct more than 70% of the variable sites within the T/F sequence for all participants for whom >20 unique proviral sequences were sampled during ART. The evolutionary history catalogued within proviruses persisting during ART can be leveraged to reconstruct T/F sequences, yielding insights into HIV evolutionary dynamics.

Background: Approximately 50% of people living with HIV experience HIV-associated neurocognitive disorders (HAND), impacting motor/cognitive functions. Peroxisome proliferator-activated receptor gamma (PPARγ), primarily involved in regulating glucose/lipid metabolism, are also known to be expressed in the brain and can elicit anti-inflammatory responses in neurodegenerative diseases. HAND is associated with brain inflammation, astrocytic activation and neuronal apoptosis. Our study aimed to investigate the effect of activating PPARγ by a novel selective agonist, INT131 to attenuate HIV-induced brain inflammation, using an ecotropic HIV-1 (EcoHIV) mouse model that simulates HAND. We also investigated the expression/localization of PPARγ, and astrocytic/neuronal apoptotic markers in HIV+ human brain samples.

Methods: We used qPCR to analyze the mRNA expression of viral genes, inflammatory cytokines/chemokines, and blood-brain barrier (BBB) tight junction proteins. BBB permeability was tested using the NaF assay, 21 days post intracranial injection of saline or EcoHIV (2x108 pg/ml) in: i) uninfected mice, ii) EcoHIV-infected mice, and iii) EcoHIV-infected mice treated with INT131 (50 mg/kg/day). Additionally, immunohistochemistry on human brain tissue (cerebellum, basal ganglia, and cortex) assessed GFAP (astrocyte marker), Casp-3 (apoptosis marker), and PPARγ protein expression/location.

Results: Exposure of mice to EcoHIV significantly increased the mRNA expression of viral genes (Vif, Tat), inflammatory markers (Tnf-α, Il-1b, Il-6, Ifn-γ) and decreased BBB markers (Ocln, Cldn5, Tjp-1) in brain regions. INT131 significantly reduced the expression of viral genes and inflammatory markers and restored the expression of BBB markers and permeability in the EcoHIV mouse model. Post-mortem brain tissues from HIV+ individuals with neurocognitive impairment showed increased expression of astrocytic/apoptotic markers and reduced PPARγ expression compared to HIV- individuals.

Conclusion: Our findings suggest PPARγ as a potential novel molecular target for treating/preventing HIV-associated brain inflammation, BBB dysfunction, and HAND. INT131 effects in reversing neurocognitive deficits will be investigated in the EcoHIV mouse model through behavioral studies. (Supported by CIHR)

Background:
Within the penile microbiome, a triad of co-occurring anaerobic bacterial species (Prevotella bivia, Peptostreptococcus anaerobius, and Dialister micraerophilus) are associated with HIV seroconversion, increased pro-inflammatory cytokines, and increased local HIV target cell density. However, observational data cannot attribute causality to these bacteria in increasing foreskin inflammation and HIV risk. To study these interactions empirically, we have developed in vitro foreskin mimetic tissues for co-culture with bacteria.

Objective:
Determine if organotypic foreskin tissues support growth of facultative and strict anaerobic penile bacteria applied as monospecies and polymicrobial inoculums in physiological (microaerophilic) oxygen conditions.

Methods:
Primary foreskin fibroblasts and keratinocytes were used to generate foreskin mimetics with dermal and stratified epidermal layers. Tissues were inoculated with ~1000 CFU of either: facultative anaerobes unassociated with HIV risk (Staphylococcus epidermidis or Corynebacterium tuberculostearicum), strict anaerobes belonging to the triad (P. bivia or P. anaerobius) or a polymicrobial inoculum containing the triad species (derived from coronal sulcus swabs). Tissue-bacterial co-cultures were incubated at 37°C in 2% O2 for 5 days. Bacterial abundance (qPCR) and viability (plating) were assessed throughout co-culture.

Results:
Monospecies facultative isolates showed increased abundance (5 log-fold and 2-log fold increase for S. epidermidis and C. tuberculostearicum, respectively) and maintained viability over 5 days. Similarly, viable strict anaerobes P. bivia (3 log-fold increase) and P. anaerobius (stable abundance) were recovered from the tissue surface after 5 days of co-culture. Stable abundance and viability were also observed for all three triad species when tissues were inoculated with the polymicrobial community and cultured at 2% O2. No tissue degradation or contamination was observed.

Conclusions:
Foreskin mimetics support colonization by facultative and strict penile anaerobes from monospecies and polymicrobial inoculums in microaerophilic conditions. These findings affirm our co-culture approach, which we will use to further investigate the influence of the penile microbiome on HIV susceptibility.

Introduction: HIV-1 infected cells evade cytotoxic T lymphocytes (CTLs) recognition by downregulating cell surface major histocompatibility complex class I (MHC-I). To accomplishes this, the HIV-1 protein Nef complexes with host Src family kinases (SFKs) to phosphorylate the Zeta-chain-associated protein kinase 70 (ZAP-70) thereby initiating a signalling cascade that results in surface MHC-I endocytosis. This evasion of CTLs contributes to HIV-1 latent reservoir persistence, a primary obstacle to curing HIV-1 infection. Consequentially, the Nef:SFK interaction represents a potential therapeutic target to reverse MHC-I downregulation by HIV-1. Methods: To further elucidate the role of Nef:SFK in MHC-I downregulation, we employed a flow cytometric analysis of ZAP-70 in primary human CD4+ T cells infected with the HIV-1 transmitted founder virus CH58. Infected cells were measured for levels of ZAP-70 and specific post-translational modifications (PTM) at activating (Tyr319) and inhibitory (Tyr292) ZAP-70 tyrosine residues. Results: Infected CD4+ T cells had significantly increased total ZAP-70 and PTM of ZAP-70 pTyr319 and pTyr292. Deletion of nef significantly decreased total ZAP-70 levels and decreased pTyr319 PTM levels. Furthermore, a significant decrease in pTyr319 PTM was observed when vpu was also deleted. Infected CD4+ T cells with high levels of pTyr292 PTM harboured significantly elevated surface MHC-I. These results suggest that HIV-1 Nef increases ZAP-70 protein levels to expand the pool of ZAP-70 available for activation. Furthermore, Nef meditates the activating pTyr319 PTM with Vpu having a compensatory role when Nef is absent. We also determined that the inhibiting ZAP-70 pTyr292 PTM is associated with higher surface MHC-I and therefore may disrupt MHC-I downregulation by HIV-1. Conclusions: These findings support that HIV-1 accessory proteins promote ZAP-70 pTyr319 PTM for enhanced downregulation of surface MHC-I. Moreover, the dynamics of ZAP-70 and pTyr292 PTM levels during infection suggest concurrent novel processes effecting surface MHC-I downregulation.