CRL4^(AMBRA1) targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling

Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4^(AMBRA1) (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4^(AMBRA1) is required to disrupt the assembly and attenuate the ligase activity of human CRL5^(SOCS3) and HIV‐1 CRL5^(VIF) complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4^(AMBRA1) modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5^(SOCS3) and CRL5^(VIF), respectively. Thus, by discovering a substrate of CRL4^(AMBRA1), ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway

CRL4^(AMBRA1) targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling

Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4^(AMBRA1) (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4^(AMBRA1) is required to disrupt the assembly and attenuate the ligase activity of human CRL5^(SOCS3) and HIV‐1 CRL5^(VIF) complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4^(AMBRA1) modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5^(SOCS3) and CRL5^(VIF), respectively. Thus, by discovering a substrate of CRL4^(AMBRA1), ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Application of CRISPR-Cas9 Gene Editing for HIV Host Factor Discovery and Validation

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

Gene targeting statistics in CEM2n.

<p>Gene targeting statistics in CEM2n.</p

Mutation summary.

<p>Mutation summary.</p

Characterization of independent knockout and knockdown clones.

<p>(A) Levels of each indicated <i>A3</i> mRNA in CEM2n, <i>A3G</i>- and <i>A3F</i>-null derivatives, and CEM-SS (relative to <i>TBP</i>; mean and s.d. shown for triplicate experiments). (B) Levels of each indicated <i>A3</i> mRNA in CEM2n or <i>A3F</i>-null cells transduced with shNS, shA3D, or shA3G constructs (relative to <i>TBP</i>; mean and s.d. shown for triplicate experiments). (C) Single-cycle infectivity of Vif-deficient HIV produced in parallel in the indicated cell lines (mean and s.d. shown for p24-normalized triplicate experiments). (D) The kinetics of Vif-proficient (blue diamonds) and Vif-deficient (red squares) HIV spreading infection in the indicated cell lines. Numbers distinguish independent clones.</p