Growing evidence suggests that ribosomes selectively regulate translation of specific mRNA subsets. Here, quantitative proteomics and cryoelectron microscopy demonstrate that poxvirus infection does not alter ribosomal subunit protein (RP) composition but skews 40S rotation states and displaces the 40S head domain. Genetic knockout screens employing metabolic assays and a dual-reporter virus further identified two RPs that selectively regulate non-canonical translation of late poxvirus mRNAs, which contain unusual 5' poly(A) leaders: receptor of activated C kinase 1 (RACK1) and RPLP2. RACK1 is a component of the altered 40S head domain, while RPLP2 is a subunit of the P-stalk, wherein RPLP0 anchors two heterodimers of RPLP1 and RPLP2 to the large 60S subunit. RPLP0 was required for global translation, yet RPLP1 was dispensable, while RPLP2 was specifically required for non-canonical poxvirus protein synthesis. From these combined results, we demonstrate that poxviruses structurally customize ribosomes and become reliant upon traditionally non-essential RPs from both ribosomal subunits for efficient initiation on their late mRNAs.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as an HIV-1 capsid (CA) binding host factor to promote viral DNA integration targeting into gene-dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC)-the sub-viral machinery that carries out viral DNA integration-are unknown. To study CPSF6's role in HIV-1 PIC function, we extracted PICs from cells that are either depleted of CPSF6 or express a mutant form that cannot bind to CA. These PICs exhibited significantly lower viral DNA integration activity when compared to the control PICs. The addition of purified recombinant CPSF6 restored the integration activity of PICs extracted from the CPSF6-mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6's role in PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in integration in these cells was detected, and this reduction was not a consequence of lower reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6-mutant cells. Finally, sequencing analysis revealed that HIV-1 integration into CPSF6-mutant cell genomes was significantly redirected away from gene-dense regions of chromatin compared to the control cells. Collectively, these results suggest that the CPSF6-CA interaction promotes PIC function both in vitro and in infected cells.IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with cellular host factors. However, the molecular details of HIV-host factor interactions are not fully understood. For instance, the HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 to restrict HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes HIV-1 integration targeting into gene-dense regions of the host genome. Here, we provide evidence that CPSF6-CA interaction stimulates the activity of HIV-1 preintegration complexes (PICs). We also describe that disruption of CPSF6-CA binding in target cells significantly reduces viral DNA integration and redirects integration targeting away from gene-dense regions into regions of low transcriptional activity. These findings identify a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

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 import3-5, uncoating6, and integration site selection4,6-11. 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) complex12,13. 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.

Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. Several host restriction factors that inhibit human immunodeficiency virus type 1 (HIV-1) have been identified, but most of them are antagonized by viral proteins. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel HIV-1 restriction factor that suppresses the production of HIV-1 and other retroviruses, but does not appear to be directly antagonized by viral proteins. It acts by binding to Gag nucleocapsid (GagNC) via zinc-finger motifs, which inhibits viral genome recruitment and results in genome-deficient virion production. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. This distinct, dual-acting antiviral mechanism makes upregulation of ZCCHC3 a novel potential therapeutic strategy.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Carcinoma dissemination can occur when heterogeneous tumor and tumor-stromal cell clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system mimicking native breast tumor microenvironment, we developed 3D computational models, which hypothesize that leader cells need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance at the leading edge. From single-cell sequencing analyses, we find that leader cells are heterogeneous and identify and isolate a keratin 14-and cadherin-3-positive sub -population sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through local production of laminin, which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration.

Carcinoma dissemination can occur when heterogeneous tumor and tumor stromal cells clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and interact with the microenvironment to direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system that mimics the native breast tumor microenvironment, we developed 3D computational models which hypothesize that leader cells generate high protrusive forces and overcome extracellular matrix (ECM) resistance. Using single cell sequencing, we reveal leader cells are heterogeneous, and identify and isolate a unique Cadherin-3 (Cdh3) positive leader cell subpopulation that is necessary and sufficient to lead migration. Cdh3 controls leader cell protrusion dynamics through the local production of Laminin-332 which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration. Teaser Higher protrusions of Cdh3+ leader cells polarize tumor organoids that then invade collagen via Lam332 adhesion feedback.

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.

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.