The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.

Human Immunodeficiency Virus (HIV) interacts with a wide array of host factors at each stage of its lifecycle to facilitate replication and circumvent the immune response. Identification and characterization of these host factors is critical for elucidating the mechanism of viral replication and for developing next-generation HIV-1 therapeutic and curative strategies. Recent advances in CRISPR-Cas9-based genome engineering approaches have provided researchers with an assortment of new, valuable tools for host factor discovery and interrogation. Genome-wide screening in a variety of in vitro cell models has helped define the critical host factors that play a role in various cellular and biological contexts. Targeted manipulation of specific host factors by CRISPR-Cas9-mediated gene knock-out, overexpression, and/or directed repair have furthermore allowed for target validation in primary cell models and mechanistic inquiry through hypothesis-based testing. In this review, we summarize several CRISPR-based screening strategies for the identification of HIV-1 host factors and highlight how CRISPR-Cas9 approaches have been used to elucidate the molecular mechanisms of viral replication and host response. Finally, we examine promising new technologies in the CRISPR field and how these may be applied to address critical questions in HIV-1 biology going forward.

Human immunodeficiency virus (HIV) relies upon a broad array of host factors in order to replicate and evade the host antiviral response. Cleavage and polyadenylation specificity factor 6 (CPSF6) is one such host factor that is recruited by incoming HIV-1 cores to regulate trafficking, nuclear import, uncoating, and integration site selection. Despite these well-described roles, the impact of CPSF6 perturbation on HIV-1 infectivity varies considerably by cell type. Here, we report that CPSF6 knock-out in primary CD4+ T cells leads to increased permissivity to HIV-1 infection due to broad transcriptional reprogramming. Knock-out of CPSF6 results in widespread differential gene expression, including downregulation of genes involved in the innate immune response and enhanced expression of the HIV-1 co-receptors. Accordingly, these cells are less responsive to interferon and express lower levels of antiretroviral restriction factors, including TRIM5α. These transcriptional changes are linked to global shortening of mRNA 3’ untranslated regions (UTRs) through changes in alternative polyadenylation (APA), which are triggered by disruption of the CPSF6-containing Cleavage Factor Im (CFIm) complex. Furthermore, we find that recruitment of CPSF6 by HIV-1 cores is sufficient to perturb CPSF6 function, leading to 3’ UTR shortening and subsequent transcriptional rewiring. These results suggest a model in which HIV-1 transcriptionally reprograms target cells through recruitment of CPSF6 to incoming cores to circumvent the antiviral response and enhance permissivity to infection.

Human immunodeficiency virus (HIV) relies upon a broad array of host factors in order to replicate and evade the host antiviral response¹. Cleavage and polyadenylation specificity factor 6 (CPSF6) is one such host factor that is recruited by incoming HIV-1 cores to regulate trafficking², nuclear import^3-5, uncoating⁶, and integration site selection^4,6-11. Despite these well-described roles, the impact of CPSF6 perturbation on HIV-1 infectivity varies considerably by cell type. Here, we report that CPSF6 knock-out in primary CD4+ T cells leads to increased permissivity to HIV-1 infection due to broad transcriptional reprogramming. Knock-out of CPSF6 results in widespread differential gene expression, including downregulation of genes involved in the innate immune response and enhanced expression of the HIV-1 co-receptors. Accordingly, these cells are less responsive to interferon and express lower levels of antiretroviral restriction factors, including TRIM5α. These transcriptional changes are linked to global shortening of mRNA 3’ untranslated regions (UTRs) through alternative polyadenylation (APA), which is triggered by disruption of the CPSF6-containing Cleavage Factor Im (CFIm) complex^12,13. Furthermore, we find that recruitment of CPSF6 by HIV-1 cores is sufficient to perturb CPSF6 function, leading to 3’ UTR shortening and subsequent transcriptional rewiring. These results suggest a novel mechanism by which HIV-1 transcriptionally reprograms CD4+ T cells through recruitment of CPSF6 to circumvent the innate immune response and enhance permissivity to infection.

Viruses have developed a diverse array of mechanisms to hijack host transcriptional machinery and ensure successful viral gene expression. One such method is through manipulation of the cellular machinery required for post-transcriptional processing of host messenger RNAs (mRNAs). 3’ end processing of host mRNA requires a complex suite of proteins that function together to identify potential polyadenylation sites, cleave the pre-mRNA at the selected site, and synthesize the polyadenosine tail. Under certain cellular conditions - including stress, disease, and infection - altered regulation of these complexes can lead to changes in alternative polyadenylation (APA) site usage, resulting in changes in 3’ untranslated region (UTR) length, transcript abundance, and translation potential. Recent studies have identified APA as an emerging regulator of viral infection. Viruses interact with polyadenylation machinery both directly and indirectly to facilitate viral gene expression, evade innate immune responses, and achieve targeted host shutoff. While the specific interactions vary, viral manipulation of 3’ post-transcriptional processing proteins is common among a range of viruses, including herpes simplex virus (HSV), influenza A virus (IAV), and human immunodeficiency virus (HIV). In this review, we provide an overview of cellular polyadenylation machinery and mechanisms of APA that are exploited by viruses, as well as the methods that can be used to analyze changes in APA. We highlight the ways in which a range of both DNA and RNA viruses manipulate post-transcriptional processing and APA to regulate viral and host gene expression and enhance cellular permissivity to infection. Finally, we highlight current knowledge gaps and future directions exploring the translatability of these discoveries toward the development of future therapeutic strategies.