Complete sequence of the Sendai virus NP gene from a cloned insert

A DNA molecule representing all but the three terminal bases of the Sendai virus nucleoprotein (NP) gene, copied from viral mRNA, was inserted into pBR322. The NP insert comprised 1673 bases. The first AUG protein initiation codon, at position 65, began an open reading frame of 1551 bases, encoding a protein of 517 amino acids with an amino acid composition corresponding to previously published data. The NP gene sequence determined in the present work is similar to that described by Shioda et al. [ Nucl . Acids Res. 11, 7317 (1983)], but there are 14 amino acid differences that probably reflect differences in virus strains. The predicted secondary structure of the NP molecule and the locations within that structure of potential protease cleavage sites are in accord with structural domains previously defined by controlled protease digestion.     

Rescue system for measles virus from cloned cDNA driven by vaccinia virus Lister vaccine strain

A rescue system for measles virus from cloned cDNA was established using CHO/hSLAM cells (Chinese hamster ovary cells expressing a measles virus receptor, signaling lymphocyte activation molecule), LO-T7-1 virus (the Lister vaccine strain of vaccinia virus expressing the T7 RNA polymerase under the control of the early/late p7.5 promoter), and caspase inhibitor. LO-T7-1 drove efficiently the T7 promoter in CHO/hSLAM cells. Rescue efficiency with LO-T7-1 was not as high as that with the vTF7-3 strain based on a neurovirulent vaccinia virus, but was increased by using a caspase inhibitor to block apoptosis of CHO/hSLAM cells induced by LO-T7-1. These modifications resulted in a measles virus rescue efficiency that was even higher than that of previous systems. This safer and more efficient system will facilitate further the genetic manipulation of measles virus in basic research and virus vector development.     

Intracellular accumulation of Punta Toro virus glycoproteins expressed from cloned cDNA

The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NSM. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NSM sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.     

An improved method for recovery of secretory immunoglobulins and lymphocytes from the nasal mucosa

The difficulty of obtaining adequate specimens for assay has severely restricted in vivo investigations of local immune responses in humans. Washing the posterior nasopharynx for an extended period using chilled saline to stimulate serous secretions has improved yields of both secretory immunoglobulins and functionally competent immunocytes. The proportions of T- and B-cells found in such washings appear to differ from those found in blood.     

The role of host responses in the recovery of mice from Sendai virus infection

The antiviral responses in mice to intranasal inoculation with Sendai virus are described. To investigate the relative importance of the humoral, cell-mediated and interferon responses, the pathogenesis of this infection was studied in animals which were immunocompetent, T cell-deprived or immunosuppressed with cyclophosphamide. Treatment with cyclophosphamide converted the mild, self-limiting infection observed in immunocompetent mice into a severe and frequently lethal pneumonic disease. This was associated with an enhanced interferon response but no detectable antibody or cell-mediated immune response. T cell-deprived mice suffer an infection of intermediate severity associated with an increased interferon response, a normal humoral immune response and no cell-mediated immune response. The implications of these results in relation to the role of the antiviral responses in recovery from Sendai virus infection are discussed.     

Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes

The Sendai virus ribonucleoprotein (RNP) showed only very low plaque-forming titers upon transfection and the virus yields after one-step growth were quite limited. We tried to enhance the Sendai virus yield by supplying the viral L and P/C gene products through vaccinia vectors. A combination of the recombinant vaccinia viruses carrying the L gene (Vac-HL) and the P/C gene (Vac-HPC), both of which were driven by the promoter of the vaccinia virus 7.5K protein gene, enhanced the yield only a little whereas another combination of Vac-HLd7.5, the L gene insert of which was driven by the promoter of the vaccinia virus thymidine kinase gene in place of the 7.5K promoter, and Vac-HPC greatly enhanced the Sendai virus yield. This seemed to correlate with the fact that the Vac-HL interfered with Sendai virus growth markedly while the Vac-HLd7.5 did not. These results strongly suggest that the L and P/C gene products act in cooperation as the RNA polymerase, and overproduction of the L protein is inhibitory for Sendai virus growth. This system seems to be of value as a tool for analyzing the functions of L and P/C genes of Sendai virus.     

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA. Starting with genomic negative sense (?) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low. Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (?)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (?)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5′ ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein. Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (?)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.     

Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs

A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells.     

Efficient rescue of measles virus from cloned cDNA using SLAM-expressing Chinese hamster ovary cells

We here report a highly efficient reverse genetics system for measles virus (MeV), using Chinese hamster ovary cells constitutively expressing a MeV receptor human signaling lymphocyte activation molecule (CHO/hSLAM cells). The recombinant vaccinia virus vTF7-3 that encodes the T7 RNA polymerase under the control of the early/late promoter was used in the system. Replication of vTF7-3 was highly restricted in CHO/hSLAM cells, but the virus could still drive the T7 promoter, allowing us to recover MeV from the transfected cDNA efficiently. With this system the number of infectious centers, in which MeV replication cycles are initiated from transfected cDNAs, was approximately 100 times higher than that with the previous system (. J. Virol. 74, 6643-6647), and the recovery rate was 100%. The wild-type MeV that encodes the lac-Z gene of approximately 3.2kb in length, was easily generated with this CHO/hSLAM system, while such virus could not be recovered with the previous system. Since SLAM acts as a cellular receptor for both MeV vaccine and wild-type strains, the Edmonston vaccine strain was also recovered with this system more efficiently than with any other systems reported previously. Thus, the CHO/hSLAM-based system would expand applications of the MeV reverse genetics by allowing productions of mutant MeVs that have been difficult to generate with less efficient systems.     

Double-layered membrane vesicles released from mammalian cells infected with Sendai virus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M.