CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

308

Monitoring the Infection of a New Strain of Simian Betaretrovirus in Southern Muriquis From Brazil Thamiris S Miranda 1 , Pedro H. Carneiro 1 , Marcus Vinicius de Mattos Silva 1 , Ronaldo da Silva Mohana Borges 1 , Silvia B. Moreira 2 , Alcides Pissinatti 2 , Orlando da Costa Ferreira Júnior 1 , Marcelo A. Soares 1 , André Felipe Andrade dos Santos 1 1 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Centro de Primatologia do Rio de Janeiro, Rio de Janeiro, Brazil Background: Neotropical primates are natural hosts of several viruses, but many are poorly studied. One example was the identification through massive sequencing of a new strain of simian betaretrovirus (SRVbar) in an individual (2506) of Southern Muriqui (Brachyteles arachnoides), a critically endangered species, who died of immunodeficiency at the Primatology Center of Rio de Janeiro (CPRJ). The phylogenetic analysis showed that the SRVbar grouped close to the SRV of Asian primates, which also causes clinical signs similar to those presented by Muriqui. Four muriquis from the same enclosure as 2506 were also positive for SRVbar, but were asymptomatic. Objective: The objective of the study was to monitor the infection of SRVbar in specimen 2506 and in asymptomatic contacts. Methods: Oral swab and blood samples were collected from five muriquis in CPRJ between 2016 and 2020. Nucleic acids were extracted from plasma, peripheral blood mononuclear cells (PBMC) and oral swab and a qPCR was performed for the env to analyze the proviral and viral loads from SRVbar. For serological analysis, a recombinant SRVbar p27Gag protein produced on the Escherichia coli cell platform was synthesized and purified by affinity chromatography to be used as a viral antigen in the Western Blot. Results: Results: The proviral load in the PBMC was 10x higher than in the oral swab of all animals tested (p=0.0001). Specimen 2506 showed similar proviral DNA loads in PBMC and oral swab 10x higher (log 8.12/10 6 cells and 7.66 copies/10 6 cells, respectively) than compared to asymptomatic animals (log 7.66 copies /10 6 cells and 6.57 copies/10 6 cells, respectively) (p=0.038). All asymptomatic animals had detectable viral loads and the average was 2.34 log 10 copies/mL of plasma (1.4 - 3.18, min-max) while 2506 had a viral load greater than 3.96 log 10 copies/mL of plasma (p=0.044). There was success in the production of the recombinant SRVbar p27Gag protein and seropositivity for this antigen varied between animals over time, where for two animals (2506 and 3078) there was an inverse relationship with viral load, as already described in Asian primates of the genus Macaca infected with SRV. Conclusion: For the first time, a study on the course of betaretrovirus infection is being investigated in Neotropical primates, contributing to the knowledge of this new viral strain. Species-Specific APOBEC3 Splicing and Variations in Viral Mutagenesis Azad Khosh 1 , Rachael Springman-Rodriguez 1 , Armando Mendez 1 , Kathryn Jackson-Jones 2 , Margarita Rzhetskaya 2 , Judd F. Hultquist 2 , Diako Ebrahimi 1 1 University of Texas at San Antonio, San Antonio, TX, USA, 2 Northwestern University, Chicago, IL, USA Background: APOBEC3 (A3) proteins are known for their antiviral activities, acting as restriction factors to directly mutate viral genomes or block different stages of viral replication. Among the seven A3 enzymes in humans, A3G is the most restrictive enzyme, unique in its ability to cause GG>AG mutations that can generate stop codons. While HIV-1 Vif counteracts A3 enzymes by targeting them for ubiquitination and proteasomal degradation, G>A mutations are still common in clinical isolates, and sublethal levels of A3-induced mutations contribute to viral diversification, drug resistance, and immune evasion in vivo. Methods: Non-human primate models are often used to study these various aspects of HIV biology even though intrinsic antiviral immunity differences between human and nonhuman primate models at a molecular level are not well characterized. A study by Zhang et al (PMID: 31776266) began to address this gap through the identification of an aberrant mRNA splicing pattern in pig-tailed macaque A3G, which is associated with a reduced level of GG>AG mutations in SIV sequences obtained from these animals. To better understand the source, prevalence, and consequence of this A3G mRNA splicing defect, we analyzed all (>250K) publicly available HIV-1 and SIV sequences as well as >100 short-read RNAseq datasets and a recent long-read RNAseq dataset from different human populations and a wide range of nonhuman primates. Results: Our investigation revealed that this splicing defect is present in many Old-World monkeys but not in Great Apes and New-World monkeys, and that the mutational signatures of SIV reflect this immune defect. Contrary to the WITHDRAWN

Methods: HIV-1 ∆env luciferase reporter viruses were pseudotyped with either HIV-1 envelope glycoprotein (Env), murine leukemia virus (MLV) Env, vesicular stomatitis virus G glycoprotein (VSV G), SARS-CoV spike (S) or SARS-CoV-2 S glycoprotein and the effect of producer-cell GBP5 expression on particle infectivity was determined. The effect of GBP5 expression on viral glycoprotein glycosylation was determined by treating virus- producing cell lysates with N-glycosidase F (PNGase F), which removes N-linked oligosaccharides from glycoproteins. Results: We found that GBP5 reduces the infectivity of particles bearing each of the viral glycoproteins tested in a concentration-dependent manner. Western blot analysis demonstrated that GBP5 causes a dose-dependent shift in the electrophoretic mobility of the viral glycoproteins. Moreover, GBP5 strongly reduced glycoprotein incorporation into virions while increasing virion associated levels of the uncleaved glycoprotein precursors. PNGase F treatment abolished the GBP5-mediated shift in the electrophoretic mobility of the glycoproteins, indicating that GBP5 affects N-linked protein glycosylation and glycan modification. Conclusion: Our data establish that GBP5 impairs viral infectivity by interfering with glycoprotein function. Furthermore, we provide evidence that GBP5 not only inhibits furin cleavage of viral glycoproteins but also affects their glycosylation regardless of whether they undergo furin-dependent processing. Moreover, our data on VSV G indicate that GBP5 targets the glycosylation of proteins other than class I fusion proteins. These results provide novel insights into the broad antagonism of viral glycoprotein function by the cellular host innate immune response. In the ongoing studies, we are testing the ability of GBP5 to restrict the replication of human and murine pathogens in mouse models, thereby providing the first insights into GBP5 antiviral activity in vivo . Retinoic Acid Blunts the Aryl Hydrocarbon Receptor/SAMHD1-Dependent HIV-1 Restriction in Macrophages Ramon Edwin Caballero 1 , Jonathan Dias 1 , Jean-Philippe Goulet 2 , Jean-Pierre Routy 3 , Andrew Mouland 4 , Petronela Ancuta 1 1 Centre de Recherche du CHUM, Montreal, Canada, 2 CellCarta, Montreal, Canada, 3 McGill University Health Centre Research Institute, Montreal, Canada, 4 McGill University, Montreal, Canada Background: As part of the cell-autonomous innate immune system, SAMHD1 restricts HIV-1 replication by limiting the pool of deoxyribonucleoside triphosphate required for efficient reverse transcription. The antiviral activity of SAMHD1 is abrogated by phosphorylation. Among regulators of SAMHD1 activity, ligands of aryl hydrocarbon receptor (AhR) were reported to decrease SAMHD1 phosphorylation via mechanisms involving the transcriptional repression of kinases CDK1/2. Conversely, we demonstrated that retinoic acid (RA) promotes HIV-1 replication in macrophages by inducing SAMHD1 phosphorylation. Considering the role of macrophages in HIV-1 infection in anatomic sites rich in both AhR ligands and RA, such as the intestine, here we investigated how the crosstalk between these two pathways governs SAMHD1 activity and HIV-1 replication in macrophages. Methods: Monocyte-derived macrophages (MDM) generated in the presence of M-CSF were exposed to all trans RA (ATRA) and/or AhR agonist (FICZ) and exposed to replication-competent or single-round VSV-G- pseudotyped HIV-1. HIV replication was measured by ELISA, FACS, and nested real-time PCR using specific primers for early/late reverse transcripts and integrated HIV-DNA. RNA Sequencing was performed using the Illumina technology. Validations were performed by Western Blot, RT-PCR or FACS. Results: While exposure to FICZ inhibited HIV-1 replication in MDM at the level of reverse transcription, integration, and translation, this antiviral effect of was absent in the presence of ATRA, which significantly increased HIV-1 replication via CCR5-dependent entry and post-entry mechanisms. FICZ decreased SAMHD1 phosphorylation in the absence but not in the presence of ATRA, consistent with the robust RA-mediated increase in CDK1 expression. Similarly, the insulin induced gene 1 (INSIG1), involved in Gag metabolism, was induced by FICZ only in the absence of ATRA. The analysis of differentially expressed genes in ATRA-treated MDM revealed the downregulation of the AhR nuclear translocator (ARNT) and other AhR target genes, and the upregulation of the gene for AhR repressor (AHRR), indicating a blunted AhR signaling pathway Conclusion: Our results demonstrate opposing roles for AhR and RA pathways in the modulation of the antiviral activity of SAMHD1 and reveal a blunted AhR-mediated antiviral program in MDMs in the presence of RA. These studies identify the need for natural SAMHD1 modulators as novel therapeutic targets for HIV-1.

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CROI 2024