C-terminus Of VP1 Research Articles

Mutational Analysis of the Carboxyl-terminal Region of the SV40 Major Capsid Protein VP1

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha'', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha''-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.

Defining residues involved in human rhinovirus 2A proteinase substrate recognition

The 2A proteinase (2A pro) of human rhinoviruses (HRVs) initiates proteolytic processing by cleaving between the C-terminus of VP1 and its own N-terminus. It subsequently cleaves the host protein eIF4GI. HRV2 and HRV14 2A pro cleave at IITTA ∗ GPSD and DIKSY ∗ GLGP on their respective polyproteins. The HRV2 2A pro cleavage site on eIF4GI is TLSTR ∗ GPPR. We show that HRV2 2A pro can self-process at the eIF4GI cleavage sequence whereas HRV14 2A pro cannot, due to the presence of the arginine residue at P1. The mutations A104C or A104S in HRV14 2A pro restored cleavage when arginine was present at P1, although not to wild-type levels. These experiments define residues which determine substrate recognition in rhinoviral 2A pro.

Analysis of minimal sequences on JC virus VP1 required for capsid assembly.

Human JC virus (JCV) belongs to the family of Polyomaviridae. The viral capsid is composed of 72 capsomeres. Five VP1 molecules make up a capsomere structure. To investigate the minimal sequences on JCV VP1 polypeptide required for capsid assembly, the first 12 (Delta N12) and 19 (Delta N19) amino acids at the N-terminus and the last 16 (Delta C16), 17 (Delta C17), and 31 (Delta C31) amino acids at the C-terminus of VP1 were truncated and expressed in E. coli. The VP1 proteins of Delta N12 and Delta C16 were able to self-assemble into a virus-like particle similar to that of wild-type (WT) VP1. However, the mutant proteins of Delta N19, Delta C17, and Delta C31 formed a pentameric capsomere structure as demonstrated by a 10-50% sucrose gradient centrifugation and electron microscopy. These results suggest that the 12 amino-terminal and 16 carboxy-terminal amino acids of VP1 are dispensable for the formation of virus-like particles, and further truncation at either end of VP1 leads to the loss of this property.

Antigenic Sites of Coxsackievirus A9

Antigenic analysis of coxsackievirus A9 (CAV9) was carried out by using a peptide scanning method. Immunogenic regions in the capsid proteins VP1, VP2, and VP3 were recognized by antibodies in the sera of virus-immunized rabbits. The peptide sequences were scanned using a 12-amino-acid window and three-residue shift. Three immunogenic regions, located in the N- and C-terminal parts of VP1 and in the N-terminus of VP3, were identified. Trypsin treatment of the virus, known to cleave off the C-terminus of VP1 containing a functional RGD motif, completely abolished the reactivity against this region but did not have any other significant effect on antigenicity. In further studies, it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sides of the motif. New antigenic sites emerged after heat treatment of CAV9 at 56 or 100°C prior to immunization; in particular, loop structures between β strands in VP2 exhibited increased immunogenicity. New antigenic sites in VP1 and VP3 also appeared after the treatments. In spite of the markedly altered reactivity in peptide scanning, the virus treated at 56°C elicited high titers of neutralizing antibodies. To reveal cross-reactive antigenic sites, antisera raised against coxsackievirus B3 and echovirus 11 were also tested. The cross-reactive antigenic sites were located mainly in the N-terminal parts of VP1 and VP3.

The Structure of a Highly Virulent Theiler's Murine Encephalomyelitis Virus (GDVII) and Implications for Determinants of Viral Persistence

GDVII is a highly virulent Theiler's murine encephalomyelitis virus (TMEV) which causes acute encephalitis in mice, while the BeAn and DA strains are the less virulent TMEV which cause chronic demyelinating disease in the central nervous system as a result of persistent infection. Purified GDVII virus isolated from infected BHK-21 cells was crystallized and its structure was determined to 3.5-Å resolution by X-ray crystallography. In contrast to other TMEV structures, the VP1 C-terminus of GDVII virus has an ordered conformation that forms a hook over the VP3 knob near the threefold axis. Comparisons with the atomic structures of the less virulent BeAn and DA viruses revealed significant structural variations in a major site (cluster B) on the protruding surface loop puff B of VP2. Puff B is located near the VP3 GH loop region which is structurally analogous to the host receptor attachment site of the major serogroup of human rhinoviruses. Mutations at residue 1101 in VP1 and residue 2141 in VP2, which are also near the VP3 GH loop and adjacent to cluster B, were previously shown to influence persistence of DA virus. These observations indicate that the characteristic interaction with the host receptor through these sites may potentially alter TMEV persistence.

Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and-mouth disease virus, defined by neutralization-resistant variants

Five neutralizing monoclonal antibodies (nMAbs) obtained against type A5 Spain-86 foot-and-mouth disease virus were used to generate a series of neutralization-resistant variants. In vitro and in vivo assays showed that the variants were fully refractory to neutralization by the selecting nMAb. On the basis of cross-neutralization and binding assays, two neutralizing antigenic sites have been located on the virus surface; one, located near the C-terminus of VP1, displayed a linear epitope, and the second, located on VP2, displayed two conformational epitopes. Nucleotide sequencing of RNA of the parental and variant capsid protein-coding region P1 has placed the amino acid changes at position 198 of VP1 for the first site and at positions 72 and 79 of VP2 for the related epitopes in the second site. The relative importance of these two sites in the biological properties of foot-and-mouth disease virus is discussed.

Substrate requirements of a human rhinoviral 2A proteinase

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P, site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P′ region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.

Principal and subsidiary antigenic sites of VP1 involved in the neutralization of poliovirus type 3.

The characterization of over 300 mutants, derived from two strains of poliovirus type 3 and selected for resistance to neutralization by monoclonal antibodies, has led to the further definition of the major antigenic site involved in neutralization. The site encompasses amino acids 89 to 100 of VP1. A subsidiary antigenic site near the C-terminus of VP1 has been identified for the Sabin vaccine strain of poliovirus type 3. Of 59 monoclonal antibodies to poliovirus type 3 examined, 27 had virus-neutralizing activity and 25 of these were identified as directed against the major site on VP1 (designated site 1), indicating the immunodominant role of this site. One of the six monoclonal antibodies that recognized the subsidiary antigenic site on VP1 (designated site 2) possessed virus-neutralizing activity. The identification of the principal antigenic site of the virus provides a rational basis for attempts at the development of synthetic oligopeptide vaccines against poliovirus type 3.

Altered virion proteins of a temperature-sensitive mutant of polyoma virus, ts59

The ts59 mutant of polyoma virus is blocked in a late step of infection at the restrictive temperature. Cellular and viral DNA synthesis proceed normally in ts59-infected cells at the restrictive temperature, but infectious progency virus particles are not assembled. The ts59 mutant complements early tsA mutants in mixed infection, and the temperature-sensitive mutation maps in the late region of the polyoma genome. The infectivity of ts59 virions is much heat labile than wild-type polyoma. All three nonhistone capsid proteins of ts59, VP1 (45,000 daltons) and the overlapping proteins VP2 (30,000 daltons) and VP3 (20,000 daltons), show altered mobilities when analyzed by SDS-polyacrylamide gel electrophoresis. The tryptic peptide patterns of all three ts59 virion proteins also differ from the tryptic peptide patterns of wild-type proteins. Analysis of the ts59 proteins synthesized in vitro and in infected cells suggests that the alterations in the ts59 virion proteins are caused by differences in primary structure rather than by post-translational modifications. The capsid proteins of convertant virions produced by marker rescue of the ts59 temperature-sensitive mutation, using various restriction endonuclease fragments of wild-type DNA, have been analyzed. Results of these studies suggest that (i) 26 map units is the furthest point, in a clockwise direction on the genetic map, that the information for the C-terminus of VP1 can be from the Eco·R1 cleavage site; (ii) the N-terminal end of VP2 extends beyond the N-terminal end of VP3; (iii) the temperature-sensitive phenotype of ts59 is correlated with a peptide alteration common to VP2 and VP3. The ts59 mutant contains two further peptide alterations not related to the temperature-sensitive phenotype: a C-terminal alteration in VP1 and an alteration unique to VP2. Cells infected by ts59 contain approximately fourfold lower amounts of viral capsid proteins and virus-specific messenger RNA at the restrictive temperature compared to the permissive temperature.