CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: These results indicate that complementation occuring between drug sensitive and drug resistant HIV is one mechanismwhich can drive and maintain an HIV quasispecies and account for the presence of drug sensitive virus in the face of ART. 180 DISRUPTION OF A RNA SECONDARY STRUCTURE IN HIV-1 gp41 INDUCES VIRAL LETHALITY Ana Jordan-Paiz 1 , Maria Nevot 1 , Kevin Lamkiewicz 2 , Marie Lataretu 2 , Sandra Franco 1 , Manja Marz 2 , Miguel Angel Martinez 1 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 2 Friedrich-Schiller University of Jena, Jena, Germany Background: Synonymous genome recoding has been widely used to study different aspects of virus biology. Previous studies have demonstrated HIV-1 attenuation by reduction in protein expression after synonymous recoding. We aim here to explore the impact of synonymous codon usage on HIV-1 Env expression and virus replication capacity. Methods: The codons AGG, GAG, CCT, ACT, CTC and GGG of HIV-1 env gene were synonymously changed to CGT, GAA, CCG, ACG, TTA and GGA, respectively. Different recoded envs were generated. Viral replication and viability was measured after transfection in MT-4 cells by quantifying HIV-1 p24 antigen production. Replication capacity assays were performed in MT-4 cells and PBMCs. WT, recoded env genes and HIV-1 rev were cloned in an expression vector (pcDNA3.1). Env expression plasmids were cotransfected with Rev expression plasmid in 293T cells. Immunoblot analyses and qPCR were performed to quantify protein expression and Env mRNA production. RNA secondary structures were obtained using Vienna RNA package. Results: A recoded env variant containing 39 mutations was lethal for the virus. WB analysis of Env expression revealed that protein expression of the recoded variant was highly reduced. To further study the mutations responsible for this phenotype, newmutants were designed by reverting substitutions to WT or reducing the number of newly generated CpG dinucleotides. Most of the new virus variants were viable, although they showed different replication capacities. Interestingly, one variant that only reverted two nucleotides that belong to the same codon showed indistinguishable replication capacity when compared to WT. Moreover, after transfection, other virus variants generated compensatory mutations next to this codon or reverted this codon to WT. Computational analyses revealed a severe disruption in a RNA secondary structure of variants containing this mutated codon. Importantly, the disrupted RNA structure was restore when this codon was reverted to WT or new mutations were introduced in the proximity. Conclusion: We show here that codon usage of the HIV-1 env strongly impact the replication capacity of the virus. Moreover, synonymous recoding of HIV-1 env gene has identified, in the gp41 coding region, an evolutionary conserved local RNA secondary structure that may be essential for virus viability. Disruption of this structure leads to severe reduction in mRNA translation and virus replication capacity. Ashokkumar Manickam 1 , Aanand Sonawanne 1 , Shambhu G. Aralaguppe 2 , Srikanth Tripathy 1 , Ujjwal Neogi 2 , Luke Elizabeth Hanna 1 1 National Institute for Research in TB, Chennai, India, 2 Karolinska Institute, Stockholm, Sweden Background: Among the repertoire of transmitted viral variants, only a small proportion of the viruses (transmitted founder (TF) viruses) are successful in establishing infection. It is widely believed that the early immune response to HIV infection is likely to be an essential factor in determining the clinical course of the disease. Thus, a better understanding of the characteristics of TF viruses and their role in early infection will throw light on the features that bestow these variants with the unique advantage of successfully establishing infection, and contribute significantly to the design and development of a protective HIV vaccine. Methods: Patient-derived 250 envelope glycoprotein, gp120 were cloned in pMN-K7-Luc-IRESs-NefDgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently infected with HIV through mother-to-child transmission and two adults who acquired infection through the heterosexual route and were in the chronic stage of the infection. 65 out of 250 clones tested were found infectious and analyzed for genetic identity and 181 GENETIC IDENTITY AND BIOLOGICAL PHENOTYPE OF EARLY TRANSMITTED FOUNDER HIV-1 VIRUSES

a block including the ARM and second OD, or the NES did not show changes in functional activity. However, exchanging a block including the n-terminus and the first OD with four amino acid changes (N-OD) was sufficient to determine activity, such that a 9-G N-OD in an 8-G background was as active as unmodified 9-G Rev (p=0.55), and vice versa. A single variation at position 24 was tested as this has been shown to affect activity in NL4-3. The 9-G Q24R mutant had a 50% reduction in activity (p=0.001) but the 8-G R24Q mutant did not show increased activity, demonstrating an additional role for the other three amino acid changes. Conclusion: The large difference in Rev-RRE activity between these primary isolates is due to four amino acid changes. Some of these residues have been implicated in Rev monomer stabilization while others may affect dimer-dimer interaction. Rev activity changes in another lentivirus are associated with clinical disease progression. Activity variation in HIV Rev may also play a role in clinical disease, such as in the establishment of latency.

Poster Abstracts

179 COMPLEMENTATION MAINTAINS QUASISPECIES OF DRUG-SENSITIVE AND -RESISTANT HIV WITH ART Laurelle A. Jackson 1 , Sandile Cele 1 , Tulio de Oliveira 2 , Jennifer Giandhari 2 , Gila J. Lustig 1 , Fabio Zanini 3 , Richard Neher 3 , Alex Sigal 1 1 Africa Health Research Institute, Mtubatuba, South Africa, 2 University of KwaZulu- Natal, Durban, South Africa, 3 University of Basel, Basel, Switzerland Background: HIV exists as multiple genotypes in a single infected individual, referred to as a quasispecies. How such sequence heterogeneity can be maintained in the same infection environment remains unclear. Methods: We reproduced a quasispecies in vitro by using the antiretroviral drug efavirenz (EFV) as the selective pressure. The cell culture infection was performed over 18 days, with fresh uninfected cells and EFV replenished every two days. We determined the frequency of drug resistance mutations to EFV using deep sequencing both at the population and single cell level. For single cell sequencing of HIV DNA we sorted the infected cells into wells of a multi-well plate at 1 cell per well, then lysed the cells to extract DNA and amplified the reverse transcriptase region. Results: We observed that while the frequency of genotypically EFV resistant virus increased with time, it never completely supplanted the drug sensitive genotype. Instead, the drug sensitive HIV genotype stabilized at approximately 20 percent of the total population. Single-cell sequencing of viral genotypes showed that the fraction of drug resistant virus in the population increased when most of the cells were infected with drug sensitive HIV but plateaued when cells were co-infected with drug sensitive and drug resistant genotypes. This suggested that in co-infected cells, drug sensitive virus may package drug resistant reverse transcriptase and become phenotypically resistant to the drug, known as phenotypic mixing or complementation. To verify that complementation can result in phenotypic drug resistance of EFV sensitive HIV, we transfected cells with CFP labelled wildtype and YFP labelled EFV resistant mutant HIV molecular viral clones, or co-transfected both. HIV from singly transfected cells followed the expected resistance pattern. However, virus from co-transfected cells was similarly resistant to EFV regardless of whether its genotype was drug sensitive or resistant.

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CROI 2020