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内容記述 |
Roles of viral NS and M proteins on the influenza virus growth were<br /> examined. The NS proteins of influenza virus, NS1I and NS2, are encoded<br /> by RNA segment 8. The NSI proitein is encoded by a colinear mRNA<br /> transcript, whereas thc NS2 protein with the molecular weight of 14.2<br /> kilodaltons (kDa) is synthesid after splicing of NSI mRNA. Up to now,<br /> these NS proteins are believed to exist only in virus-infected cells. The NSl<br /> protein, which localizes in nuclei of virus-infected cells, recognizes the <i>cis</i>-<br />acting sequence on NSI mRNA and controls its splicing to NS2 mRNA.<br /> NS2 is also present mainly in the nuclei. At present, however, little is<br /> known on the function of this protein. This study indicates that the NS2<br /> protein, previously considered as one of the two nonstructural proteins (NS 1<br /> and NS2), exists in virus particles as a structural component. By<br /> immunochemical method, the number of NS2 molecules in a virus particle<br /> was estimated to be 130-200 molecules. After solubilization of viral<br /> enve]ope, NS2 was still associated with ribonucleoprotein (RNP) cores, but<br /> was later dissociated from RNP upon removal of the membrane M1 protein.<br /> A filter-binding assay <i>in vitro</i> indlicated direct protein-protein contact<br /> between M1 ancl NS2. Following chemical cleavage of the M1 protein,<br /> NS2 was found to bincl only a C-terminal fragment of M1. By an<br /> immunoprecipitation method, NS2-M1 complexes were also detected in<br /> virus-infected cel] lysates. These observations altogether indicate specific<br /> molecu]ar nteraction between M1 and NS2, suggestlng that NS2 regulates<br /> the function of M1 or vice versa. <br /> The M gene of influenza viruses encodes 2 proteins, M1 and M2.<br /> The M 1 protein is tightly associated with virions forming a matrix, which<br /> associates with RNP at its internal surface but interacts with envelope at its<br /> external surface. M1 interacts with both NP, thereby interferes with the<br /> function of RNP-associated RNA polymerase, and NS2 in virions. In<br /> virus-infected cells, M1 is involved in both early (uncoating and import of<br /> RNP into infectecl nuc]ei) and late (assembly of virions during maturation<br /> and export of RNP from nuclei into cytoplasm) stages of virus growth. On<br /> the othcr hand, M2 forms an ion channel and is considered to control the<br /> transport of hemagglutinin (HA). M2 may also control uncoating step to<br /> release RNP in the early phase of virus infection. Genetic studies described<br /> in this report suggested that one or both of the M proteins have a regulatory<br /> role(s) of the rate of virus growth. Influenza virus A/WSN/33 forms large<br /> plaques (>3mm diameter) on MDCK cells whereas A/Aichi/2/68 forms<br /> on]y small plaques (<1mm diameter). Fast growing reassortants (AWM),<br /> isolated by mixed infection of MDCK cells with these two virus strains in<br /> the presence of anti-WSN antibodies, all carried the M gene from WSN.<br /> On MDCK cells, these reassortants produced progeny viruses as rapidly as<br /> did WSN, and the virus yield was as high as Aichi. Pulse-labeling<br /> experiments at various times after virus infectlon showed that the reassortant<br /> AWM started to synthesize viral proteins earlier than Aichi. To determine<br /> which of the two M proteins, M1 or M2, is responsible for the fast rate of<br /> virus growth, an attempt was made to make recombinant viruses possessing<br /> the chimeric M gene between WSN and Aichi. For this purpose, I<br /> employed a newly developed RNA-transfection method into helper virus-<br />infected cells. The ts-mutant derived from WSN, ts5 1 , carrying the ts lesion<br /> only in thc M gene, were used as a helper virus to rescue the chimeric M<br /> gene RNA. A transfectant virus carrying a chimeric M gene consisting of<br /> WSN-MI and Aichi-M2, CWA20, was generated by using an improved<br /> reverse genetics system. The CWA20 virus formed large-sized plaques,<br /> indicating that thc M1 protein, but not the M2 protein, was responsible for<br /> this rapid growth of WSN-type. Taken together, I conclude that the<br /> reassortant viruses entry into growth cycle faster than the parent Aichi strain,<br /> presumably due to rapid uncoating of the M1 protein from RNP cores. As a<br /> result of rapid uncoating, the reassortant RNP should be transported into<br /> host nuclei faster than Aichi RNP, ultimately leading to an early onset of<br /> transcription of the viral genes. |
要旨・審査要旨 / Abstract, Screening Result (284.7 kB)
