Katsuhiro Kiyotani

The paramyxovirus, Sendai virus, V protein encodes a luxury function required for viral pathogenesis

The Sendai virus (SeV) V protein is characterized by the unique cysteine‐rich domain in its carboxy‐terminal half which is fused to the amino‐terminal half of the P protein, but its function has remained enigmatic. The V protein‐directing mRNA is generated by a remarkable process known as mRNA editing involving the pseudotemplated addition of a single G residue at a specific septinucleotide locus in the P gene, whereas the unedited exact copy encodes the P protein. Here, we introduced two nucleotide changes in the septinucleotide motif (UUUUCCC to UUCUUCC) in a full‐length SeV cDNA and were able to recover a virus from the cDNA, which was devoid of mRNA editing and hence unable to synthesize the V protein. Compared with the parental wild‐type virus with regard to gene expression, replication and cytopathogenicity in various cell lines in vitro, the V(−) virus was found to be either potentiated or comparable but never attenuated. The V(−) virus, however, showed markedly attenuated in vivo replication capacity in and pathogenicity for mice. Thus, though categorized as a non‐essential gene product, SeV V protein encodes a luxury function required for in vivo pathogenicity.

Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C′, C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several‐fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C‐like proteins, but their roles also remain to be defined.

Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication

Sendai virus (SeV) is an enveloped virus with a negative sense genome RNA of about 15.3 kb. We previously established a system to recover an infectious virus entirely from SeV cDNA and illustrated the feasibility of using SeV as a novel expression vector. Here, we have attempted to insert a series of foreign genes into SeV of different lengths to learn how far SeV can accommodate extra genes and how the length of inserted genes affects viral replication in cells cultured in vitro and in the natural host, mice. We show that a gene up to 3.2 kb can be inserted and efficiently expressed and that the replication speed as well as the final virus titers in cell culture are proportionally reduced as the inserted gene length increases. In vivo, such a size‐dependent effect was not very clear but a remarkably attenuated replication and pathogenicity were generally seen. Our data further confirmed reinforcement of foreign gene expression in vitro from the V(−) version of SeV in which the accessory V gene had been knocked out. Based on these results, we discuss the utility of SeV vector in terms of both efficiency and safety.

AIP1/Alix is a binding partner of sendai virus C protein and facilitates virus budding

ABSTRACT The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.

Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

ABSTRACT The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C341S and V-C365R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV VΔC, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

Immediate protection of mice from lethal wild-type Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, possessing homologous interfering capacity.

Protection of mice from lethal Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, which was isolated from a carrier culture, was studied. HVJpi had a strong interfering capacity with the replication of virulent wild-type virus in LLCMK2 cells. When a high dose of HVJpi (3.0 x 10(7) CIU) was inoculated intranasally into mice, the mice showed neither illness nor lung lesions but gained significant resistance against the challenge of virulent wild-type virus (18 LD50) immediately after inoculation. In contrast, the mice inoculated with a lower dose of HVJpi (8.2 x 10(5) CIU) did not show the immediate resistance but became immune several days after inoculation. Time courses of the virus replication in the lung revealed that the replication of wild-type virus was strongly suppressed to about 1/1000 by the simultaneous infection with a high dose of HVJpi, thus resulting in minimizing the lung lesions and survival of all the mice infected. Neither interferon nor natural killer cells appeared to play a major role in the immediate immune status by HVJpi, since no difference was observed in protection of mice simultaneously infected with wild-type virus and HVJpi in spite of pretreatment of the mice with anti-interferon and anti-asialo GM1 antibodies as compared with that of the untreated doubly infected mice. On the other hand, it was suggested by analysis of viral polypeptides synthesized in the lung of infected mice by Western blotting that the early stage of replication of wild-type virus in the lung was inhibited mainly by the interfering capacity of HVJpi. These results indicate that HVJpi is an unique virus mutant which is capable of protecting mice from lethal Sendai virus infections by its interfering capacity immediately after inoculation and then by the induction of virus-specific immune responses.

Inhibition of Interferon Regulatory Factor 3 Activation by Paramyxovirus V Protein

ABSTRACT The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferon regulatory factor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhibited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is important as an alternative target of paramyxovirus V proteins.

Importance of the Anti-Interferon Capacity of Sendai Virus C Protein for Pathogenicity in Mice

ABSTRACT The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K151A, E153A, and R154A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K151A, E153K, and R157L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1−/− mice but was cleared from those of STAT1+/+ mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.

Conserved and Non-Conserved Regions in the Sendai Virus Genome: Evolution of a Gene Possessing Overlapping Reading Frames

We have sequenced the entire genome of a virulent field isolate of Sendai virus, the Hamamatsu strain, and compared the sequence with that of a distant related strain, the Z strain. Calculation of synonymous and non-synonymous (amino acid changing) nucleotide substitutions revealed regions where changes were permissive and non-permissive, and the experimentally determined functional region were found to be conserved, showing that important regions for function were conserved during evolution. In the cistron-overlapping regions in the P gene, one reading frame was conserved, whereas the other overlapping frame was flexible. The priority of one frame could be a strategy for evolution of an overlapping gene of RNA viruses. We found that the carboxyl two thirds of the C protein was conserved over the amino-terminal one third, possessing priority to the overlapping P polypeptide. This suggests that the carboxyl two thirds of the C protein have a functional importance. We also found a highly variable region between the L coding frame and the 5′ trailer sequence. The relevance of these findings to actual viral replication should be clarified in the future.

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin‐like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the −4, −2 and −1 positions upstream of the cleavage site were essential, and that at the −5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the −5, −2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.