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It is shown that reovirus capsid inner layer consists of 132 capsomeres with a skewed icosahedral pattern of T = 13 (left). 780 structural units composing capsomeres are integrated into trimers. The boundary between capsomeres runs between the structural units forming trimers.
Capsid polypeptides of all six types (B1-6) of group B coxsackieviruses were compared by high-resolution gel electrophoresis, and synthesis of protein and RNA in B4- or B5-infected HeLA cells was analyzed. Four polypeptides, VP1-4, were detected in each type. Another polypeptide, VP0, slightly larger than VP1, was also detected in trace amounts in some types. VP1-3 showed different but characteristic molecular weights (VP1, 34,500 to 37,000; VP2, 31,000 to 36,000; VP3, 26,000 to 32,500), and presented well-defined and reproducible differences in electrophoretic mobility. The molecular weight of VP4 ranged from 5,000 to 5,500. VP1 was largest in B2 and B4, smallest in B1, and of intermediate size in the other types. VP2 was largest in B4 and smallest in B2; VP3 was largest in B5 and B6 and smallest in B4. In B4- or B5-infected HeLa cells, host protein synthesis began to decline after 2 hours postinfection and was less than 20 percent of the control by 6 hours postinfection. Actinomycin D-resistant viral RNA synthesis started at about 2 hours postinfection, peaked by 5 hours, and then declined rapidly. Virus-specific protein synthesis began while host protein synthesis was declining, increased during the ensuing period, and declined in late infection. A number of virus-specific proteins with molecular weights from 23,500 to greater than 92,500 were detected in the host cytoplasm. At least three of these proteins were also present in the nucleus. The kinetics pf processing of virus-specific proteins were examined by pulse-chase experiments in B5-infected cells. The relative intensities of [35S]-methionine-labeled polypeptides suggest that a number of smaller, stable chains (MW 23,500 to 38,000) are generated by cleavage of a precursor polypeptide (MW 92,500 to 100,000).
Inactivation of poliovirus at subfreezing temperature in the presence of unusually low concentrations of urea (less than or equal to 0.5 M) was investigated. Whereas serotypes 1 and 2 are very sensitive, type 3 is resistant. Inactivation cannot be attributed to concentration of solutes since temperature must be reduced below -13 degrees C for loss of infectivity. Characteristics of the inactivated virion are similar to those of virions in the early stages of uncoating in HeLa cells, viz., loss of infectivity, sensitivity to proteases and detergents, change in isoelectric point, retention of intact genome, and in some instances, loss of VP4. The molecular basis for inactivation is considered to be dissociation of water bound to capsid proteins thereby causing irreversible denaturation of native tertiary structure. The results of this study are discussed in terms of their relevance to the early stages of uncoating in vivo.
Western blot analysis of several dasheen mosaic virus isolates revealed molecular weight differences among their capsid proteins (CPs). Sequence analysis of the CP gene showed that the CP of isolate TEN was 15 amino acids shorter than that of isolate LA due to a 57-base deletion and a 12-base insertion into the 5' end of the TEN CP gene. Our data suggest that frequent deletions and insertions are responsible for the CP size diversity among DMV isolates.
Monoclonal IgG to the inner capsid of human rotavirus was produced by mice immunized with hybrid cells derived from mouse myeloma and the spleens of mice immunized with rotavirus. This IgG reacted with an antigenic determinant of mammalian rotaviruses that was not present on that of avian rotaviruses.
The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSVS8 is 1914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1810 which is capable of encoding a protein of M(r) 67,348. The N-terminal amino acid sequence of a approximately 43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a approximately 43K major capsid protein.
The DNA complementary to the 3'-terminal 1 404 nucleotides [excluding the poly(A) tail] of papaya leaf-distortion mosaic potyvirus (PLDMV) RNA was cloned and sequenced. The sequence starts within a long open reading frame (ORF) of 1 195 nucleotides and is followed by a 3' non-coding region of 209 nucleotides. Capsid protein (CP) is encoded at the 3' terminus of the ORF. The CP contains 293 residues and has a Mr of 33 277. The CP of PLDMV exhibits 49 to 59% sequence similarity at the amino acid level to the CPs of papaya ringspot potyvirus (PRSV) and other potyviruses. This result is consistent with the absence of a serological relationship between PLDMV and PRSV or other potyviruses. The results support the assignment of PLDMV as a distinct member of the genus Potyvirus.
A 401bp DNA fragment of ferret Aleutian disease virus (ADV) was amplified using PCR primers spanning a hypervariable region of mink ADV capsid sequence. The amplified fragment was 88-89% homologous to the same region of previously known sequence of three different strains of mink ADV, however, as low as 54% homology was observed when compared with a 39bp segment known as hypervariable region. Within the predicted 13 amino acid hypervariable region, the ferret ADV sequence differed at 6 positions from the wild type mink Utah1 strain. Three amino acids Gln289, Glu293 and Thr295 in this region were common to the pathogenic ferret ADV, mink Utah1 and ADVK strains, but differed from the cell culture adapted nonpathogenic mink strain ADVG suggesting that these three conserved residues may have some functional significance.
Hepatitis A virus (HAV) was propagated in a hepatoma cell line and complete viral particles with a density of 1.34 g/ml were purified from cell extracts. The topography of the viral proteins (VPs) was studied by surface labelling with 125I and a solid-phase oxidant. The order of labelling intensity in complete particles was VP1 much greater than VP3 greater than VP2; labelling of VP4 was undetectable. When the particles were denatured with sodium dodecyl sulfate at 100 degrees C before iodination, the labelling efficiency was 6 times higher and the order of labelling intensity was VP3 greater than VP2 greater than VP1. After denaturation, the viral proteins no longer reacted with human anti-HAV antibody. The results suggest that (i) as with other picornaviruses, HAV exposes an essential part of VP1 at its surface whereas VP3 and especially VP2 are more hidden; (ii) naturally immunized individuals do not form detectable amounts of antibodies against the denatured capsid proteins. The apparent molecular weights of the VPs were 33000, 29000 and 28000 daltons.
Eight new sequences of European isolates from almond, apple, hop, prune and pear of the Apple mosaic ilarvirus (ApMV) capsid protein gene are presented. A consensus sequence was established as having 654 nucleotides (nt) and two American and two European isolates were identified to have insertions 6 to 15 nucleotides after nt position 141. The insertion resulted in the American isolate A inframeshift repaired with two point insertions 17 and 68 nt downstream. The RNA around the insertion point can potentially form a stable secondary structure with three hairpins. The insertions could stabilise this structure or could be neutral. The predicted folding of the translated protein is not influenced by the insertions or frameshift, and we speculate that the region after nt position 141 is without reasonable selection pressure and represents a hot spot for the accumulation of insertion mutations in ApMV.
Tropical iridovirus infection causes severe epizootic resulting in mass mortalities and large economic losses in freshwater ornamental fishes cultured in Southeast Asian countries, in wild fish seedlings captured in South China Sea, and in marine fishes farmed in Japan, Singapore, and Thailand. All of tropical iridovirus-infected fishes histopathologically showed the systemic formation of inclusion body-bearing cells and necrosis of virus-infected splenocytes and hematopoietic cells. We designed primer sets for the ATPase gene and the major capsid protein (MCP) gene and sequenced the PCR products derived from 5 iridovirus isolates from sea bass in South China Sea, red sea bream in Japan, brown-spotted grouper with a grouper sleepy disease in Thailand, dwarf gourami from Malaysia and African lampeye from Sumatra Island, Indonesia. The ATPase gene and the MCP gene of these 5 viral isolates were highly homologous (> 95.8%, > 94.9% identity, respectively) and the deduced amino acid sequences of the ATPase and the MCP were also highly identical (> 98.1%, > 97.2% identity, respectively). Based on the high homology, these 5 isolates of tropical iridovirus from various fishes in geographically different regions were determined to have a single origin and to be native to Southeast Asian regions. However, these sequences were far different from those of members of the genera Ranavirus, Lymphocystivirus and Iridovirus in the Family Iridoviridae. We propose a new genus "Tropivirus" for tropical iridovirus in the Family Iridoviridae.
Dragon grouper, Epinephelus lanceolatus, nervous necrosis virus (DGNNV) consists of 180 copies of capsid protein that encapsulates a bipartite genome of single-stranded (+)RNAs. Expressing the open reading frame (ORF) of RNA2 in Escherichia coli forms virus-like particles (VLPs) that resembles native virus. Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively.
We have analyzed isolates of two recently circulating genotypes (genotypes VI and VII) of type A foot-and-mouth disease virus (FMDV) from India. Maximum-likelihood models provided support for the presence of positively selected sites in the capsid-coding (P1) region. Positive selection was detected at a number of amino acid positions behind a background of strong purifying selection. Among the positively selected sites, four were identified at known critical antigenic residues (VP2 79, VP3 59 and 70 and, VP1 83), suggesting that FMDVs are under pressure from the immune system. Two residues (VP2 134 and VP3 59) that are part of the heparan sulfate-binding pocket in subtype A22 FMDV are also inferred to be under positive selection. Antigenic divergence was observed between and within the genotypes in neutralization tests with sera raised against the representative isolates from genotypes VI and VII. The two vaccine strains showed one-way antigenic relationships (r value) of <0.2 with 64% of the isolates, whereas, with genotypes VI and VII an r value of >0.4 was observed with 24% and 64% of the isolates, respectively. No correlation could be deduced from the amino acid substitutions at specific critical residues and lower r values in the field isolates.
The production of vaccine antigens in plants is a safe and potentially very cost-effective alternative to traditional expression systems. We investigated the possibility of transgenic plant expression of the Human papillomavirus (HPV) type 16 L1 major capsid protein, with and without nuclear localisation signals, in Nicotiana tabacum cv. Xanthi plants. The genes were stably integrated into the N. tabacum genome, and both the expressed proteins were capable of assembling into capsomers and virus-like particles. The proteins in concentrated leaf extracts (L1(Tr)) were tested for antigenicity using a panel of characterised monoclonal antibodies (Mabs). Neutralising and conformation-specific Mabs (H16:V5 and H16:E70) were shown to bind to both types of the plant-produced particles. We estimated the L1(Tr) product yield to be 2-4 microg per kg of fresh leaf material. Rabbits immunised with small doses of plant-produced particles elicited a weak anti-HPV-16 L1 immune response. Our results support the feasibility of using transgenic plants for the production of HPV vaccines.
Ten iridoviruses were isolated from cultured fish from various regions in Korea; 7 from rock bream, 1 from red sea bream, 1 from sea bass, and 1 from rockfish. The full open-reading frame (ORF) encoding the major capsid protein (MCP) (1362 bp) from ten iridoviruses were sequenced and the nucleotide sequences were phylogenetically analyzed. Phylogenetic analysis revealed that the ten Korean isolates were classified into one cluster. However, their sequences were not identical and, based on the nucleotide sequence variation, they could be further divided into two subgroups. While nine Korean isolates were similar to the Japanese isolate red sea bream iridovirus (RSIV), one isolate was distinct from other iridovirus isolates. These results suggest that a diversity of iridoviruses exist in Korea and that a new variant strain has emerged.
Bohle iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.
Genome segment 10 (S10) of Threadfin aquareovirus (TFV) was cloned, sequenced, analyzed and found to be 987 bp long encoding a protein of 298 aa with a predicted molecular mass of 32.0 kDa. The TFV S10 gene possesses terminal motifs, (5' GTTTTA and ATTCATC 3') which are also conserved in the S6 and S11 TFV gene segments. Sequence comparison revealed that the TFV S10 gene was similar to the Striped bass reovirus (SBR) VP7 outer capsid protein (OCP). A conserved putative zinc-finger motif, CCHC, present in the mammalian reovirus (MRV) delta3 protein, was identified in TFV and other aquareovirus VP7 protein. Phylogenetic analysis of the TFV VP7 protein indicated that TFV is closely related to SBR and Chum salmon reovirus (CSV) and possibly belong to the same species Aquareovirus A as SBR and CSV. The TFV VP7 protein was expressed in E. coli, purified and injected into mice. Serum specific antibodies were generated, however, the serum showed weak neutralizing activity. In contrast, co-incubation of this serum with another serum obtained from mice immunized with another OCP encoded by the TFV S6 gene segment resulted in a highly elevated antibody neutralization titer.
Immunoblot analysis of apple latent spherical cheravirus (ALSV)-infected leaves using a polyclonal antibody against the 21 C-terminal amino acids of a 53 K/42 K movement protein (MP) showed that a protein with an Mr of 42 kDa (42KP) is the dominant form found in vivo, which could indicate that the second AUG is used as an initiation codon of a ORF in RNA2. Co-expression of GFP with 42KP in tobacco epidermal cells showed that 42KP is able to facilitate cell-to-cell trafficking of GFP that is expressed in the same cells. The analysis of deletion mutants on each of MP, Vp24, Vp20, or Vp25 using an ALSV vector that stably expresses GFP indicated that an MP and three capsid proteins are all indispensable for the cell-to-cell movement of the virus. In ultrathin sections of infected leaves, a file of virus-like particles passing through the plasmodesmata connecting neighboring cells and tubular structures containing virus-like particles extending into the cytoplasm were observed. These results show that ALSV moves from cell to cell as virus particles.