Belete Ayele Desimmie

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), continues to wreak havoc, threatening the public health services and imposing economic collapse worldwide. Tailoring public health responses to the SARS-CoV-2 pandemic depends on understanding the mechanism of viral replication, disease pathogenesis, accurately identifying acute infections, and mapping the spreading risk of hotspots across the globe. However, effective identification and isolation of persons with asymptomatic and mild SARS-CoV-2 infections remain the major obstacles to efforts in controlling the SARS-CoV-2 spread and hence the pandemic. Understanding the mechanism of persistent viral shedding, reinfection, and the post-acute sequalae of SARS-CoV-2 infection (PASC) is crucial in our efforts to combat the pandemic and provide better care and rehabilitation to survivors. Here, we present a living literature review (January 2020 through 15 March 2021) on SARS-CoV-2 viral persistence, reinfection, and PASC. We also highlight potential areas of research to uncover putative links between viral persistence, intra-host evolution, host immune status, and protective immunity to guide and direct future basic science and clinical research priorities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) continues to wreak havoc threatening the public health services and imposing economic collapse worldwide. Tailoring public health responses to the SARS-CoV-2 pandemic depends on understanding the mechanism of viral replication, disease pathogenesis, and accurately identifying acute infections and mapping the spreading risk of hotspots across the globe. However, effective identification and isolation of persons with asymptomatic and mild SARS-CoV-2 infections remain the major obstacles to efforts in controlling the SARS-CoV-2 spread and hence the pandemic. Understanding the mechanism of persistent viral shedding, reinfection, and the post-acute sequalae of SARS-CoV-2 infection (PASC) is crucial in our efforts to combat the pandemic and provide better care and rehabilitation to survivors. Here we present a living literature review on SARS-CoV-2 viral persistence, reinfection and PASC. We also highlight potential areas of research to uncover putative links between viral persistence, intra-host evolution, host immune status, and protective immunity to guide and direct future basic science and clinical research priorities.

Mammals have developed clever adaptive and innate immune defense mechanisms to protect against invading bacterial and viral pathogens. Human innate immunity is continuously evolving to expand the repertoire of restriction factors and one such family of intrinsic restriction factors is the APOBEC3 (A3) family of cytidine deaminases. The coordinated expression of seven members of the A3 family of cytidine deaminases provides intrinsic immunity against numerous foreign infectious agents and protects the host from exogenous retroviruses and endogenous retroelements. Four members of the A3 proteins—A3G, A3F, A3H, and A3D—restrict HIV-1 in the absence of virion infectivity factor (Vif); their incorporation into progeny virions is a prerequisite for cytidine deaminase-dependent and -independent activities that inhibit viral replication in the host target cell. HIV-1 encodes Vif, an accessory protein that antagonizes A3 proteins by targeting them for polyubiquitination and subsequent proteasomal degradation in the virus producing cells. In this review, we summarize our current understanding of the role of human A3 proteins as barriers against HIV-1 infection, how Vif overcomes their antiviral activity, and highlight recent structural and functional insights into A3-mediated restriction of lentiviruses.

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFβ) at 3.9-Å resolution. The structure shows that Vif and CBFβ form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFβ beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis—for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.

HIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif–CBFβ interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFβ, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif–CBFβ interactions that are important for Vif function has not been performed. Here, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFβ and their ability to induce A3G degradation as well as rescue HIV-1 replication in the presence of A3G. We found that multiple Vif residues are involved in the extensive N-terminal Vif–CBFβ interaction and that the 5WQVMIVW11 region of Vif is the major determinant. A minimum of three alanine substitutions are required to completely abrogate the Vif–CBFβ interaction and Vif’s ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFβ revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction with W5 of Vif to maintain a stable and functional Vif–CBFβ complex. We also determined that CBFβ amino acids 73WQGEQR78, which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif. Our results provide detailed insight into the Vif–CBFβ interactions that are critical for Vif function and may contribute to the rational design of HIV-1 inhibitors that block Vif-mediated degradation of A3 proteins.

Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence of A3G + A3F and A3G + A3H, exhibited extensive comutation of viral cDNA, as determined by the frequency of G-to-A mutations in the proviral genomes in the contexts of A3G (GG-to-AG) and A3D, A3F or A3H (GA-to-AA) edited sites. The copackaging of A3G + A3F and A3G + A3H resulted in an additive increase and a modest synergistic increase (1.8-fold) in the frequency of GA-to-AA mutations, respectively. We also identified distinct editing site trinucleotide sequence contexts for each APOBEC3 protein and used them to show that hypermutation of proviral DNAs from seven patients was induced by A3G, A3F (or A3H), A3D and A3G + A3F (or A3H). These results indicate that APOBEC3 proteins can be copackaged and can comutate the same genomes, and can cooperate to inhibit HIV replication.