Kentaro Fujita

Replication of poliovirus requires binding of the poly(rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site.

ABSTRACT The 5′ nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CDpro. The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C93-95 and C98-100) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C93-95) resulted in slower viral growth, whereas the C98-100A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C94-95) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5′-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CDpro to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.

The nonstructural proteins of Nipah virus play a key role in pathogenicity in experimentally infected animals.

Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V−), rNiV(C−), and rNiV(W−), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V−) and rNiV(C−) were lower than the other recombinants. The rNiV(V−), rNiV(C−) and rNiV(W−) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V−) and rNiV(C−) but not the rNiV(W−) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.

Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA

We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N–RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C‐terminal region from the core region, phosphorylated amino acids were assayed using MALDI‐TOF/TOF and ESI‐Q‐TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with 32P demonstrated that these two sites are the major phosphorylated sites within the MV‐N protein. In transcriptional analysis using negative‐strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild‐type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.

Heparin-like glycosaminoglycans prevent the infection of measles virus in SLAM-negative cell lines

The wide tissue tropism of the measles virus (MV) suggests that it involves ubiquitously expressed molecules. We have constructed a recombinant MV expressing the enhanced green fluorescent protein (EGFP) (rMV-EGFP) and demonstrated that the rMV-EGFP infected several cell types (HEK-293, HepG2, Hep3B, Huh7, and WRL68 cells) that do not express the human signalling lymphocyte activation molecule (SLAM), which is known as a cellular receptor for morbilliviruses. MV infection of HEK-293 and HepG2 cells was not inhibited in an infectivity-inhibition assay using an anti-SLAM monoclonal antibody, indicating that MV could infect cells without using SLAM. Soluble heparin (HP) inhibited the rMV-EGFP infectivity in SLAM-negative cell lines in a dose-dependent manner. Direct interaction between purified virions and HP was detected in a surface plasmon resonance assay. We also demonstrated that the hemagglutinin (H) protein, but not the fusion (F) protein is responsible for the interaction between the virions and HP. Taken together, our results suggest that HP-like glycosaminoglycans bind to the H protein of MV and play a key role in the infection of SLAM-negative cells.

In vitro recombination of feline herpesvirus type 1

SummaryWe investigated the possibility of in vitro recombination of three different trains of feline herpesvirus type 1 (FHV-1), a virulent strain (C7805), a vaccine strain (F2), and a candidate vaccine strain with a deletion in the thymidine kinase gene (C7301dlTK). The results showed that recombination within different genotypes of FHV-1 strains could occur at a relatively high frequency ranging from 10.1% to 21.5% in vitro. These results indicate the generation of multiple mutant viruses by in vitro recombination experiments. The possibility for generating a virulent virus or novel type of FHV-1 by recombination in vivo is discussed.

Novel phosphoprotein-interacting region in Nipah virus nucleocapsid protein and its involvement in viral replication.

ABSTRACT The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.

Rinderpest virus H protein: role in determining host range in rabbits

A major molecular determinant of virus host-range is thought to be the viral protein required for cell attachment. We used a recombinant strain of Rinderpest virus (RPV) to examine the role of this protein in determining the ability of RPV to replicate in rabbits. The recombinant was based on the RBOK vaccine strain, which is avirulent in rabbits, carrying the haemagglutinin (H) protein gene from the lapinized RPV (RPV-L) strain, which is pathogenic in rabbits. The recombinant virus (rRPV-lapH) was rescued from a cDNA of the RBOK strain in which the H gene was replaced with that from the RPV-L strain. The recombinant grew at a rate equivalent to the RPV-RBOK parental virus in B95a cells but at a lower rate than RPV-L. The H gene swap did not affect the ability of the RBOK virus to act as a vaccine to protect cattle against virulent RPV challenge. Rabbits inoculated with RPV-L became feverish, showed a decrease in body weight gain and leukopenia. High virus titres and histopathological lesions in the lymphoid tissues were also observed. Clinical signs of infection were never observed in rabbits inoculated with either RPV-RBOK or with rRPV-lapH; however, unlike RPV-RBOK, both RPV-L and rRPV-lapH induced a marked antibody response in rabbits. Therefore, the H protein plays an important role in allowing infection to occur in rabbits but other viral proteins are clearly required for full RPV pathogenicity to be manifest in this species.

Seroepidemiological survey of distemper virus infection in the Caspian Sea and in Lake Baikal

Forty Caspian seals were surveyed seroepidemiologically between 1993 and 1998 around the times of mass mortality that occurred in 1997 in the Caspian Sea and seven Baikal seals were also surveyed in 1998. Virus neutralizing tests and ELISA clearly suggested that distemper virus epidemic was caused in Caspian seals before the spring of 1997 and that CDV infection continued to occur in Lake Baikal in recent years.

Vaccine efficacy of recombinant feline herpesvirus type 1 expressing immunogenic proteins of feline calicivirus in cats

SummaryWe previously constructed a recombinant feline herpesvirus type 1 (FHV1), C7301dlTK-Cap, which contains an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain in the deleted locus of the thymidine kinase (TK) deficient mutant (C7301dlTK) of FHV 1. In this report, we carried out in vivo experiments to assess the vaccine efficacy of the recombinant C7301dlTK-Cap against FCV and FHV 1 infections in cats. As a result, two vaccinations with the C7301dlTK-Cap by intraocular, intranasal and oral routes protected cats to a significant degree against subsequent virulent challenges with both parent FCV F4 and FHV 1 C7301 strains. The results are applicable for the further development of a new genetically engineered polyvalent vaccine for cats.

Pathological and phylogenetic features of prevalent canine distemper viruses in wild masked palm civets in Japan

Ten wild masked palm civets infected with canine distemper virus (CDV), captured in Japan from 2005 to 2007, were histopathologically and phylogenetically analyzed. Phylogenetic analysis based on the amino acid sequences of the H protein of two CDV isolates from masked palm civets revealed that the two isolates were classified into the clade of recent isolates in Japan. Histopathologically marked lesions of virus encephalitis were present in the brain, whereas gastrointestinal lesions were absent or at a mild degree. The distribution of the lesions resembles that of recent CDV cases in dogs. Therefore, recent CDV infections in masked palm civets could be caused by recently prevalent CDV in dogs. The possibility of the masked palm civet as a spreader of CDV among wildlife is also discussed.