scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. STEDMAN
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1 ◽  
Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. Stedman
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Capsid Protein Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Parvovirus LuIII transducing vectors packaged by LuIII versus FPV capsid proteins: the VP1 N-terminal region is not a major determinant of human cell permissiveness

2004 ◽  
Vol 85 (5) ◽  
pp. 1251-1257 ◽  
Author(s):  
Ian H. Maxwell ◽  
Françoise Maxwell
Keyword(s):  
Capsid Protein ◽  
Human Cell ◽  
Major Capsid Protein ◽  
Human Cells ◽  
Terminal Region ◽  
Major Determinant ◽  
Capsid Proteins ◽  
Entry Level ◽  
Capsid Protein Vp1 ◽  
Post Entry

Human cell lines are permissive for LuIII, a member of the rodent group of autonomous parvoviruses. However, LuIII vectors pseudotyped with feline panleukopaenia virus (FPV) capsid proteins can transduce feline cells but not human cells. Feline transferrin receptor (FelTfR) functions as a receptor for FPV. Transfection of Rh18A, a human rhabdomyosarcoma cell line, with FelTfR enabled transduction by vector with FPV capsid. This was not true of other human lines, suggesting restriction at some additional, post-entry, level(s) in human cells other than Rh18A. It seemed a reasonable hypothesis that a second blockage might be in nuclear delivery mediated by the N-terminal region of the minor capsid protein, VP1. We therefore generated virions containing an LuIII–luciferase genome, packaged using chimaeric VP1 molecules (N-terminal region of LuIII VP1, fused with body of FPV, and vice versa) together with the major capsid protein, VP2, of FPV or LuIII. The virions were tested for ability to transduce feline and human cells. Our hypothesis predicted that the N-terminal region of LuIII VP1 should allow transduction of human cells expressing FelTfR, while the FPV N-terminal region should not allow transduction of human cells (except for Rh18A). The experimental results did not bear out either of these predictions. Therefore, the VP1 N-terminal region appears not to be a major determinant of permissiveness for LuIII, versus FPV, capsid in human cells.

scholarly journals Major Capsid Protein Synthesis from the Genomic RNA of Feline Calicivirus

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Christian Urban ◽  
Christine Luttermann
Keyword(s):  
Capsid Protein ◽  
Viral Protein ◽  
Major Capsid Protein ◽  
Start Codon ◽  
Feline Calicivirus ◽  
Genomic Rna ◽  
Rna Translation ◽  
Vp1 Protein ◽  
Leader Protein ◽  
Positive Strand Rna

ABSTRACT Caliciviruses have a positive-strand RNA genome with a length of about 7.5 kb that contains 2, 3, or 4 functional open reading frames (ORFs). A subgenomic mRNA (sg-RNA) is transcribed in the infected cell, and both major capsid protein viral protein 1 (VP1) and minor capsid protein VP2 are translated from the sg-RNA. Translation of proteins from the genomic RNA (g-RNA) and from the sg-RNA is mediated by the RNA-linked viral protein VPg (virus protein, genome linked). Most of the calicivirus genera have translation mechanisms leading to VP1 expression from the g-RNA. VP1 is part of the polyprotein for sapoviruses, lagoviruses, and neboviruses, and a termination/reinitiation mechanism was described for noroviruses. Vesiviruses have no known mechanism for the expression of VP1 from the g-RNA, and the Vesivirus genus is the only genus of the Caliciviridae that generates VP1 via a precursor capsid leader protein (LC-VP1). Analyses of feline calicivirus (FCV) g-RNA translation showed a low level of VP1 expression with an initiation downstream of the original start codon of LC-VP1, leading to a smaller, truncated LC-VP1 (tLC-VP1) protein. Deletion and substitution analyses of the region surrounding the LC-VP1 start codon allowed the identification of sequences within the leader protein coding region of FCV that have an impact on VP1 translation frequency from the g-RNA. Introduction of such mutations into the virus showed an impact of strongly reduced tLC-VP1 levels translated from the g-RNA on viral replication. IMPORTANCE Caliciviruses are a cause of important diseases in humans and animals. It is crucial to understand the prerequisites of efficient replication of these viruses in order to develop strategies for prevention and treatment of these diseases. It was shown before that all caliciviruses except vesiviruses have established mechanisms to achieve major capsid protein (VP1) translation from the genomic RNA. Here, we show for the first time that a member of the genus Vesivirus also has a translation initiation mechanism by which a precursor protein of the VP1 protein is expressed from the genomic RNA. This finding clearly points at a functional role of the calicivirus VP1 capsid protein in early replication, and we provide experimental data supporting this hypothesis.

scholarly journals Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3CproProteinase

1999 ◽  
Vol 73 (8) ◽  
pp. 6220-6227 ◽  
Author(s):  
Annette Martin ◽  
Danièle Bénichou ◽  
Shih-Fong Chao ◽  
Lisette M. Cohen ◽  
Stanley M. Lemon
Keyword(s):  
Capsid Protein ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Recognition Sequence ◽  
C Terminus ◽  
Vaccinia Viruses ◽  
Capsid Protein Vp1 ◽  
Vp1 Capsid Protein

ABSTRACT Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the familyPicornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cproproteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cprorecognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.

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