Investigation of the Vesicular Stomatitis Virus Matrix Protein: Uncoating and Assembly

Vesicular Stomatitis Virus (VSV) is a simple, enveloped, nonsegmented negative-strand RNA virus and is the prototype rhabdovirus to study viral entry, transcription, replication, and assembly. The matrix protein (M) of VSV is a central component of the viral replication cycle. While being the smallest of the viral proteins it is multifunctional and is involved in uncoating, cytopathic effects (CPE), and assembly of the virus. M protein interactions involved in the uncoating and assembly of VSV have been examined in this dissertation. Uncoating of VSV involves dissociation of M from the ribonucleoprotein core (RNPs) of the virus. Current models of VSV uncoating propose that following membrane fusion M protein is released from the RNP with subsequent diffusion of M into the cytoplasm and distribution of some of the released M to the nucleus of a host cell. The studies in Chapter 2 investigated where in the endocytic pathway uncoating occurs, where M is located following uncoating, and the role of the cytoskeleton in distribution of input M by using a VSV, containing fluorescent M protein (rVSV-M-Lumio-Green). I found that uncoating occurs primarily in early endosomes and results in the majority of M remaining associated with the endosomal membrane which eventually localizes to the perinuclear recycling endosomes. A small fraction of M, which is presumably released into the cytosol, gets delivered to the nuclear envelope, and I found that the typical polymerized actin or microtubules within host cells were not required for distribution of M to the nuclear envelope. Uncoating and assembly of the VSV genome occurs on membranes within the cytoplasm of the host cell. Exactly how both of these processes can occur in the same environment (e.g. the cytoplasmically exposed membrane surface) without modification of M protein by phosphorylation, cleavage, or some other change has been an intriguing question in the field. In Chapter 3 I present results showing a pH effect on the M-Lumio-Green protein fluorescence in vitro and during the endocytosis of virions which was dependent on G protein. I also observed that low pH enhanced the release of M from rVSV-wt virions, which suggested that acidification of the virus interior results in the dissociation of M contacts within the virus enhancing the uncoating process. An exposed protease-sensitive loop located between amino acids 120 to 129 of M has been shown to be important for M protein self-association and has been proposed to be crucial for assembly of virions. This knowledge comes from protease treated, purified M protein and not from mutagenesis studies. In Chapter 4 I examined mutations in the exposed loop and in particular a conserved aspartate at residue 125 of a conserved LXD sequence. I found that virions with mutations at residue 123 or 125 of the LXD motif have two phenotypes; 1) an assembly defect and 2) reduced viral protein synthesis starting at 4 hours post infection. These two phenotypes have not been separated genetically and the LXD motif may represent a motif of M involved in assembly and support of VSV protein translation.Vesicular Stomatitis Virus (VSV) is a simple, enveloped, nonsegmented negative-strand RNA virus and is the prototype rhabdovirus to study viral entry, transcription, replication, and assembly. The matrix protein (M) of VSV is a central component of the viral replication cycle. While being the smallest of the viral proteins it is multifunctional and is involved in uncoating, cytopathic effects (CPE), and assembly of the virus. M protein interactions involved in the uncoating and assembly of VSV have been examined in this dissertation

Mutual Antagonism between the Ebola Virus VP35 Protein and the RIG-I Activator PACT Determines Infection Outcome

SummaryThe cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection

A Spatio-Temporal Analysis of Matrix Protein and Nucleocapsid Trafficking during Vesicular Stomatitis Virus Uncoating

To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion

Recombinant Vesicular Stomatitis Virus Vaccine Vectors Expressing Filovirus Glycoproteins Lack Neurovirulence in Nonhuman Primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use

Hemorrhagic Fever Viruses: Pathogenesis and Countermeasures

Before December 2019 and the COVID-19 pandemic, the general public was to some extent aware that zoonotic viruses can spill over into the human population and cause a disease outbreak [...

Ferret Models for Henipavirus Infection

Henipaviruses are emerging zoonotic viruses that can cause outbreaks of severe respiratory and neurological disease in humans and animals such as horses. The mechanism by which these viruses can cause disease remain largely unknown and to date there are no therapeutics or vaccines approved for use in humans. Nipah virus is listed on the World Health Organization R &amp; D Blueprint list of epidemic threats. In order to advance the availability of effective therapeutics and vaccines and medicines that can be used to save lives and avert large scale crises, animal models are required which recapitulate the disease progression in humans. Ferrets are highly susceptible to infection with henipaviruses and develop both severe respiratory and neurological disease. Therefore, the ferret model is highly suitable for studies into both the pathogenesis of henipaviruses, as well as pre-clinical evaluation of intervention strategies.</p

Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein▿

To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating

Cell fractionation and analysis of the distribution of RNPs and M protein during virus entry.

<p>rVSV-wt was bound to cells in the cold for 90 minutes in the presence of cycloheximide to prevent new protein synthesis, the inoculum was removed and the cells were washed and then either removed from the dish immediately (t-0) or warmed to 37°C and then harvested for fractionation at the times indicated. Fractions were subjected to immunoblot analysis using polyclonal anti-VSV sera. Relevant regions of the immunoblot are shown. NV (no virus) indicates cells that were mock infected and harvested at t-0. Graphs below the blots show quantification of N or M protein in each fraction. (A) N protein detected in the P16 (plasma membrane-associated virions) and NDG pellet (detergent-resistant nucleocapsids) fractions. Times post-entry are shown above the immunoblot. VSV N is a lane containing purified virus. (B) M protein detected in the S16 (plasma membrane and mitochondrial membrane) and NDG supernatant (endosomal and nuclear membrane) fraction. NV (no virus) mock infected cells. VSV M is the lane containing purified virus. (C) The four fractions probed with antibodies to the indicated cellular markers.</p

Release of RNPs from endosomes and viral protein synthesis in the presence of nocodazole.

<p>Confocal images of cells inoculated with rVSV-MLG at t-0 (A) and t-120 (B and C) post-NH₄Cl washout in the presence of cycloheximide and nocodazole (B), or just nocodazole (C), and stained for VSV N protein using N mAb conjugated to Alexa Fluor-568. Quantification of the amount of colocalization was determined for 50 individual cells using the colocalization function in the LSM software version 3.2. Percent of N protein that colocalized with MLG is indicated for t-0 and t-120 (n  =  50 cells). Bars  =  5 µm.</p