The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous ...functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.
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•RNAi and CRISPR/Cas9 screens were used to find flavivirus dependencies•The screens recovered host factors involved in endocytosis and heparin sulfation•The EMC is required by DENV and ZIKV in the early stages of replication•These studies give a systems-wide view of human-flavivirus interactions
Savidis et al. identify DENV and ZIKV dependencies using orthologous RNAi and CRISPR/Cas9 approaches. Multiple host factors involved in endocytosis and transmembrane protein processing, including the endoplasmic reticulum membrane complex (EMC), are important for flaviviral replication. Together, their studies generate a systems-wide view of human-flavivirus interactions.
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► APOBEC deaminases edit RNA and DNA. ► APOBEC regulation. ► APOBEC restrict foreign genetic elements.
APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first ...enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein–protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins.
A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH–gL–UL128–UL130–UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which ...are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT–mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.
HIV-1 integration into the host genome favors actively transcribed genes. Prior work indicated that the nuclear periphery provides the architectural basis for integration site selection, with viral ...capsid-binding host cofactor CPSF6 and viral integrase-binding cofactor LEDGF/p75 contributing to selection of individual sites. Here, by investigating the early phase of infection, we determine that HIV-1 traffics throughout the nucleus for integration. CPSF6-capsid interactions allow the virus to bypass peripheral heterochromatin and penetrate the nuclear structure for integration. Loss of interaction with CPSF6 dramatically alters virus localization toward the nuclear periphery and integration into transcriptionally repressed lamina-associated heterochromatin, while loss of LEDGF/p75 does not significantly affect intranuclear HIV-1 localization. Thus, CPSF6 serves as a master regulator of HIV-1 intranuclear localization by trafficking viral preintegration complexes away from heterochromatin at the periphery toward gene-dense chromosomal regions within the nuclear interior.
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•CA-CPSF6 interaction as opposed to nuclear periphery dictates HIV-1 integration•CPSF6 enables HIV-1 to penetrate the nuclear interior beyond the nuclear periphery•Loss of CPSF6 interaction results in integration at lamina-associated domains•LEDGF/p75 does not play a significant role in intranuclear HIV-1 localization
Prior work indicated that the nuclear periphery dictated HIV-1 integration site selection. Using multiple orthologous approaches, Achuthan et al. fail to garner evidence for preferential targeting of the periphery. The interaction between viral capsid and CPSF6 enables HIV-1 to bypass integration into peripheral heterochromatin and penetrate the nuclear structure for integration.
Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, ...we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs) at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.
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•Host proteins that modulate HRV replication were found by using MORR screens•RNASEK is needed for the replication of HRV, influenza A virus, and dengue virus•RNASEK localizes to the cell surface and endosomal pathway along with the V-ATPase•RNASEK is needed for endocytosis, and its loss produces enlarged clathrin-coated pits
Perreira et al. screened multiple orthologous RNAi reagents and identified host proteins that modulate human rhinovirus (HRV) replication. They found that RNASEK is needed for the replication of HRV, influenza A virus, and dengue virus, associates with the vacuolar ATPase (V-ATPase), and is required for endocytosis.
People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal ...treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options
. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently
. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.
APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of ...higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181-194 in the N-terminus and aa 314-320 and 345-374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15-29, 41-52 and 83-99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states.
The discovery and implementation of CRISPR/Cas9 tools in pooled genetic screens have allowed for the rapid, high-fidelity identification of host-virus interactions. However, pooled CRISPR screening ...has significant limitations in its ability both to perform cell biology and plate reader-based screens and to find alleles that result in intermediate-strength phenotypes. Here we introduce an arrayed CRISPR screening method, FACS-IT, which allows researchers to use high content imaging analysis, plate reader assays, cell supernatant characterization, and percent infectivity to characterize CRISPR-mediated gene disruptions causing both moderate and extreme phenotypic changes. By using flow sorting capabilities and CRISPR libraries that are widely available, FACS-IT overcomes both the significant limitation of pooled screening approaches and the prohibitive costs of large-scale arrayed CRISPR reagents. In doing so, FACS-IT will enable researchers to creatively use CRISPR screening to obtain a deeper understanding of biology across a wide range of fields and applications.
APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains ...unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription.
The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant ...therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.
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