Role of M Protein Aggregation in Defective Assembly of Temperature-Sensitive M Protein Mutants of Vesicular Stomatitis Virus

Self Cite

The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Virus assembly is a complex process involving a number of important steps. For enveloped viruses, the viral genome must be packaged into a nucleoprotein (nucleocapsid), which must be brought to the host membrane. This nucleocapsid must acquire a lipid bilayer by budding from the host membrane (for a review, see reference 14). For negative-strand RNA viruses of the order Mononegavirales and for the families Orthomyxoviridae and Retroviridae, these assembly functions appear to be carried out largely by viral matrix, or M, proteins (28,32). This multifunctional nature of the M protein makes it a critical player in virus assembly, which has led to a great deal of interest in determining how M proteins carry out such different functions. M proteins from different viruses have very different sequences and structures (32), a fact that has led to few initial clues about how they carry out their multiple functions and has highlighted the importance of studying individual M proteins to shed light on the wider class of functional analogs.The M protein of vesicular stomatitis virus (VSV) is a particularly well-studied example of this class of proteins. VSV M protein has been shown to play several roles in the virus assembly pathway. VSV M is responsible for condensing the viral nucleocapsid, causing the collapse of the loosely coiled, flexible nucleocapsid into the characteristic tightly coiled, bullet-like shape found in assembled virions (23,25,26). Mutagenesis and proteolysis studies have shown that the interaction of M protein with nucleocapsid requires an N-terminal sequence of the protein (2, 17). The N-terminal sequence (amino acids 1 to 50) is protease accessible in assembled nucleocapsid-M (NCM) complexes (17) and, by using a photoactivatable membrane probe, has been shown to associate with cellular membranes (20). The sequence PPPY contained within the N-terminal sequence is involved in a late stage of budding, allowing the release of budded virions from the membrane (15).In addition to the N-terminal sequence, several different regions in the middle of the protein sequence have been implicated in virus assembly, but these studies have yet to be extended to an understanding of whether these sequences cooperate and how they contribute to viral assembly and budding in an intact virus. In particular, proteolysis studies have suggested the contribution of a 4-amino-acid (aa) region at aa 121 to 124 in membrane association (9).A detailed understanding of why t...

Section: Mutagenesis and Bacterial Expression Of M Proteinmentioning

confidence: 99%

Role of Residues 121 to 124 of Vesicular Stomatitis Virus Matrix Protein in Virus Assembly and Virus-Host Interaction

Connor

McKenzie

Lyles

2006

Self Cite

“…Considered the key protein for assembly and budding, M is found mainly in the cytoplasm (80%) and sometimes is linked to the plasma membrane (10 to 20%) (51). The M protein can also be found in the nucleus (31), where it can inhibit transcription by interacting with the RNA polymerase II TFIID complex (1,6,(13)(14) and with NUP98, resulting in an inhibition of the nucleocytoplasmic transport of host mRNAs (20,34). It also inhibits cellular translation (15) by modifying the initiation complex eIF4F through dephosphorylation of the initiation factors eIF4E and 4E-BP1 (10).…”

mentioning

confidence: 99%

Mutations in the Glycoprotein of Vesicular Stomatitis Virus Affect Cytopathogenicity: Potential for Oncolytic Virotherapy

Janelle

Brassard

Lapierre

et al. 2011

Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G 6R triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G 6R inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.

“…L111 in the VSV Ind M protein is located at the alpha-helix region in which the amino acid is surrounded by hydrophobic amino acids (47). It has been suggested that the L111F mutation in the M protein of VSV Ind aggregates M protein on the cytoplasmic membrane and prevents the initiation of nucleocapsid-M protein complex formation (28) for the assembly of VSV particles at a nonpermissive temperature (39°C). Leucine is not present exactly at the same 111th position in the M protein of VSV NJ , but it is found at the 110th position.…”

Section: Discussionmentioning

confidence: 99%

“…One of the temperature-sensitive M gene mutants of the VSV Ind Orsay strain, the tsO23 mutant, has shown limited replication in a mouse glial cell line at 37°C and 39°C (27). It has been demonstrated that the temperature sensitivity of the tsO23 mutant was the result of improper assembly or lack of initiation in viral assembly, with the characteristic bullet-shaped structure at 39°C (28,29). In addition, the original tsO23 mutant lost its neurovirulence in mice, as demonstrated by direct inoculation of the virus into the brain (27).…”

Section: Esicular Stomatitis Virus (Vsv) Is a Rapidly Replicating Vmentioning

confidence: 99%

Creation of Matrix Protein Gene Variants of Two Serotypes of Vesicular Stomatitis Virus as Prime-Boost Vaccine Vectors

Kim

Hong

et al. 2015

To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSV Ind ) with a temperature-sensitive mutation (tsO23) of the VSV Ind Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSV NJ ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSV NJ (G22E M48R M51R) [rVSV NJ (GMM)] and VSV NJ (G22E M48R M51R L110F) [rVSV NJ (GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSV Ind significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK 21 cells and SH-SY5Y human neuroblastoma cells infected with rVSV Ind (GML), rVSV NJ (GMM), and rVSV NJ (GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperaturesensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSV Ind (GML) and increased stability. Twenty consecutive passages of rVSV Ind (GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene. V esicular stomatitis virus (VSV) is a rapidly replicating virus.Eventually humoral and cellular immune responses against VSV are elicited in the animal host, like any other viral vectors (1-3). Animals infected with VSV develop immune responses within 2 weeks and start to neutralize the VSV (1). This hinders the efficacy of boost immunizations for vaccination with the same vector. Serotype-specific antibodies against the viral surface glycoprotein G neutralize VSV. Two different serotypes of VSV, VSV-Indiana (VSV Ind ) and VSV-New Jersey (VSV NJ ), show 50% amino acid homologies in glycoprotein G (4). Antibodies raised against one serotype of VSV do not neutralize the other serotype of VSV (5). Accordingly, other investigators have used VSV Ind with its own surface glycoprotein G and VSV Ind carrying the G gene from other serotypes as a vaccine vector (6, 7).VSV causes self-limiting disease in animals such as pigs, cows, and horses, whereby vesicular lesions on the mouth, nose, teats, and hooves are cleared in a couple of weeks after the onset (8).Although it is very rare, human infections with VSV have been reported mostly in animal care workers, veterinarians, and laboratory personnel who were in close contact with the diseased animals or with the viruses (9-11)....