The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions

ABSTRACTBone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, theBST2gene is duplicated into two paralogs termedoBST2AandoBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the “tethering” mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A.IMPORTANCEBST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of theBST2gene calledoBST2AandoBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.

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Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2012197117 ◽

2020 ◽

Vol 117 (50) ◽

pp. 32105-32113 ◽

Cited By ~ 3

Author(s):

Ruochen Zang ◽

James Brett Case ◽

Eylan Yutuc ◽

Xiucui Ma ◽

Sheng Shen ◽

...

Keyword(s):

Membrane Fusion ◽

Vesicular Stomatitis Virus ◽

Molecular Basis ◽

Enveloped Viruses ◽

Late Endosomes ◽

Wide Range ◽

Therapeutic Development ◽

Antiviral Activities ◽

Vesicular Stomatitis ◽

Catalyzed Membrane

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

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Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Journal of Virology ◽

10.1128/jvi.01371-15 ◽

2015 ◽

Vol 89 (23) ◽

pp. 11750-11760 ◽

Cited By ~ 10

Author(s):

Timothy K. Soh ◽

Sean P. J. Whelan

Keyword(s):

Plasma Membrane ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Proteins ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Viral Particles ◽

The Matrix ◽

Vesicular Stomatitis ◽

Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

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Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus.

The Journal of Cell Biology ◽

10.1083/jcb.101.1.60 ◽

1985 ◽

Vol 101 (1) ◽

pp. 60-65 ◽

Cited By ~ 14

Author(s):

M Ono ◽

K Mifune ◽

A Yoshimura ◽

S Ohnishi ◽

M Kuwano

Keyword(s):

Vesicular Stomatitis Virus ◽

Semliki Forest Virus ◽

Ph Value ◽

Gradient Centrifugation ◽

3T3 Cells ◽

Resistant Mouse ◽

Enveloped Viruses ◽

Ph Values ◽

Percoll Gradients ◽

Vesicular Stomatitis

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.

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Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State

Journal of Virology ◽

10.1128/jvi.01372-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 5

Author(s):

Tonya Kueck ◽

Louis-Marie Bloyet ◽

Elena Cassella ◽

Trinity Zang ◽

Fabian Schmidt ◽

...

Keyword(s):

Antiviral Activity ◽

Vesicular Stomatitis Virus ◽

Rna Synthesis ◽

Inhibition Mechanism ◽

Target Sequence ◽

Messenger Rnas ◽

Antiviral Protein ◽

Viral Mrnas ◽

Antiviral State ◽

Vesicular Stomatitis

ABSTRACT Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis. IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.

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