scholarly journals A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

2021 ◽  
Author(s):  
Anthony J. Snyder ◽  
Andrew T. Abad ◽  
Pranav Danthi
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Vesicular Stomatitis Virus ◽  
Late Endosome ◽  
Host Factors ◽  
Viral Attachment ◽  
Reovirus Infection ◽  
Mammalian Orthoreovirus ◽  
Vesicular Stomatitis ◽  
Wd Repeat

ABSTRACTSuccessful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it delayed viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a broad, previously unappreciated role for WDR81 in promoting the replication of late penetrating viruses.AUTHOR SUMMARYViruses are obligate intracellular parasites that require the contributions of numerous host factors to complete the viral life cycle. Thus, the host-pathogen interaction can regulate cell death signaling and virus entry, replication, assembly, and egress. Functional genetic screens are useful tools to identify host factors that are important for establishing infection. Such information can also be used to understand cell biology. Notably, genome-scale CRISPR-Cas9 knockout screens are robust due to their specificity and the loss of host gene expression. Mammalian orthoreovirus (reovirus) is a tractable model system to investigate the pathogenesis of neurotropic and cardiotropic viruses. Using a CRISPR-Cas9 screen, we identified WD repeat-containing protein 81 (WDR81) as an essential host factor for reovirus infection of murine cells. Ablation of WDR81 blocked a late step in the viral entry pathway. Further, our work indicates that WDR81 is required for the entry of vesicular stomatitis virus that expresses the Ebolavirus glycoprotein.

scholarly journals IFN-I Independent Antiviral Immune Response to Vesicular Stomatitis Virus Challenge in Mouse Brain

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 326
Author(s):  
Anurag R. Mishra ◽  
Siddappa N. Byrareddy ◽  
Debasis Nayak
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Vesicular Stomatitis Virus ◽  
Antiviral Response ◽  
Type I ◽  
Immune Gene ◽  
Antiviral Immune Response ◽  
Virus Challenge ◽  
Vesicular Stomatitis ◽  
Ifn Γ

Type I interferon (IFN-I) plays a pivotal role during viral infection response in the central nervous system (CNS). The IFN-I can orchestrate and regulate most of the innate immune gene expression and myeloid cell dynamics following a noncytopathic virus infection. However, the role of IFN-I in the CNS against viral encephalitis is not entirely clear. Here we have implemented the combination of global differential gene expression profiling followed by bioinformatics analysis to decipher the CNS immune response in the presence and absence of the IFN-I signaling. We observed that vesicular stomatitis virus (VSV) infection induced 281 gene changes in wild-type (WT) mice primarily associated with IFN-I signaling. This was accompanied by an increase in antiviral response through leukocyte vascular patrolling and leukocyte influx along with the expression of potent antiviral factors. Surprisingly, in the absence of the IFN-I signaling (IFNAR−/− mice), a significantly higher (1357) number of genes showed differential expression compared to the WT mice. Critical candidates such as IFN-γ, CCL5, CXCL10, and IRF1, which are responsible for the recruitment of the patrolling leukocytes, are also upregulated in the absence of IFN-I signaling. The computational network analysis suggests the presence of the IFN-I independent pathway that compensates for the lack of IFN-I signaling in the brain. The analysis shows that TNF-α is connected maximally to the networked candidates, thus emerging as a key regulator of gene expression and recruitment of myeloid cells to mount antiviral action. This pathway could potentiate IFN-γ release; thereby, synergistically activating IRF1-dependent ISG expression and antiviral response.

scholarly journals Vesicular Stomatitis Virus Oncolysis of T Lymphocytes Requires Cell Cycle Entry and Translation Initiation

2008 ◽  
Vol 82 (12) ◽  
pp. 5735-5749 ◽  
Author(s):  
Stephanie Oliere ◽  
Meztli Arguello ◽  
Thibault Mesplede ◽  
Vanessa Tumilasci ◽  
Peyman Nakhaei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
T Lymphocytes ◽  
Vesicular Stomatitis Virus ◽  
Oncolytic Virus ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Activated T Cells ◽  
Cell Cycle Entry ◽  
Vesicular Stomatitis

ABSTRACT Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4+ T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G0. Activation of primary CD4+ T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH2-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G1 arrest, but not by aphidicolin or Taxol, which blocks at the G11S or G21M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G0/G1 transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G0 phase to the G1 phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.

scholarly journals Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

EMBO Reports ◽  
2009 ◽  
Vol 10 (10) ◽  
pp. 1154-1160 ◽  
Author(s):  
Papia Chakraborty ◽  
Joachim Seemann ◽  
Ram K Mishra ◽  
Jen‐Hsuan Wei ◽  
Lauren Weil ◽  
...  
Keyword(s):  
Cell Death ◽  
Vesicular Stomatitis Virus ◽  
Mitotic Progression ◽  
Vesicular Stomatitis

scholarly journals Phenotypic Consequences of Rearranging the P, M, and G Genes of Vesicular Stomatitis Virus

1999 ◽  
Vol 73 (6) ◽  
pp. 4705-4712 ◽  
Author(s):  
L. Andrew Ball ◽  
Craig R. Pringle ◽  
Brian Flanagan ◽  
Victoria P. Perepelitsa ◽  
Gail W. Wertz
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Gene Order ◽  
Gene Rearrangement ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Growth Potential ◽  
Wild Type Virus ◽  
Ancestral Gene ◽  
Vesicular Stomatitis

ABSTRACT The nonsegmented negative-strand RNA viruses (orderMononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3′ end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3′ promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.

Gene expression of vesicular stomatitis virus genome RNA

Virology ◽  
1992 ◽  
Vol 188 (2) ◽  
pp. 417-428 ◽  
Author(s):  
Amiya K. Banerjee ◽  
Sailen Barik
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Virus Genome ◽  
Vesicular Stomatitis

scholarly journals Antiviral Innate Responses Induced by VSV-EBOV Vaccination Contribute to Rapid Protection

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Andrea R. Menicucci ◽  
Allen Jankeel ◽  
Heinz Feldmann ◽  
Andrea Marzi ◽  
Ilhem Messaoudi
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Cell Activation ◽  
Ebola Virus ◽  
Virus Disease ◽  
Multiorgan Failure ◽  
Ebola Virus Disease ◽  
Phase Iii ◽  
Lethal Challenge ◽  
Vesicular Stomatitis

ABSTRACTEbola virus (EBOV) is a single-stranded RNA virus that causes Ebola virus disease (EVD), characterized by excessive inflammation, lymphocyte apoptosis, hemorrhage, and coagulation defects leading to multiorgan failure and shock. Recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (VSV-EBOV), which is highly efficacious against lethal challenge in nonhuman primates, is the only vaccine that successfully completed a phase III clinical trial. Additional studies showed VSV-EBOV provides complete and partial protection to macaques immunized 7 and 3 days before EBOV challenge, respectively. However, the mechanisms by which this live-attenuated vaccine elicits rapid protection are only partially understood. To address this, we carried out a longitudinal transcriptome analysis of host responses in whole-blood samples collected from cynomolgus macaques vaccinated with VSV-EBOV 28, 21, 14, 7, and 3 days before EBOV challenge. Our findings indicate the transcriptional response to the vaccine peaks 7 days following vaccination and contains signatures of both innate antiviral immunity as well as B-cell activation. EBOV challenge 1 week after vaccination resulted in large gene expression changes suggestive of a recall adaptive immune response 14 days postchallenge. Lastly, the timing and magnitude of innate immunity and interferon-stimulated gene expression correlated with viral burden and disease outcome in animals vaccinated 3 days before challenge.IMPORTANCEEbola virus (EBOV) is the causative agent of Ebola virus disease (EVD), a deadly disease and major public health threat worldwide. A safe and highly efficacious vesicular stomatitis virus-based vaccine against EBOV is the only platform that has successfully completed phase III clinical trials and has been used in recent and ongoing outbreaks. Earlier studies showed that antibodies are the main mode of protection when this vaccine is administered 28 days before EBOV challenge. Recently, we showed this vaccine can provide protection when administered as early as 3 days before challenge and before antibodies are detected. This study seeks to identify the mechanisms of rapid protection, which in turn will pave the way for improved vaccines and therapeutics. Additionally, this study provides insight into host gene expression signatures that could provide early biomarkers to identify infected individuals who are at highest risk of poor outcomes.

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