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December 1, 2006

Use of Reverse Genetics to Create a Variant of Sendai Virus, RGV19, with Six Amino Acid Substitutions in the Fusion Protein


Karina Palomares, Nancy L. McQueen


Wild-type Sendai virus causes a localized respiratory tract infection in mice, while a mutant, F1-R, causes a systemic infection (1).  Previous studies found two phenotypic differences between wt and F1-R Sendai viruses that we hypothesized were the critical determinants of the systemic infection caused by F1-R.  First, the fusion (F) protein of F1-R demonstrates enhanced proteolytic cleavability, which allows the virus to undergo multiple rounds of replication in multiple organs (4, 7).  Wild-type virus, on the other hand, can only undergo multiple rounds of replication in the lungs.  Sequencing of the F gene of F1-R revealed six amino acid substitutions that might contribute to the enhanced cleavability of F1-R F, including one at position 115 adjacent to the cleavage site (2).  Second, F1-R demonstrates the ability to bud from both the apical and basolateral domains of the bronchial epithelium, which allows dissemination of virus to various organs via the bloodstream (5, 6).  In contrast, wild-type virus can only bud from the apical domain, which restricts release of virus into the lumen of the respiratory tract.  Studies have shown that expression of F1-R M causes a disruption of the microtubule network of infected cells which subsequently leads to the bipolar budding exhibited by F1-R (3).  Sequencing of the matrix (M) gene of F1-R revealed two mutations that lead to amino acid substitutions, at positions 128 and 210, both of which have been shown to contribute to the microtubule disruption caused by F1-R M expression (2).

To prove our hypothesis that the mutations in the F and M genes of F1-R are the critical determinants of the systemic infection caused by  F1-R, a reverse genetics technique was used to generate viruses with various combinations of the mutations in the F and M genes of F1-R.  A reverse genetics virus that contains all six of the F1-R F mutations and both of the F1-R M mutations has recently been shown to cause a systemic infection in mice. Although we believe that the two mutations in M contribute to the pantropic phenotype of F1-R, we need to prove this by making a virus that has all of the F mutations and neither of the M mutations. Thus, the overall objective of the current study is to create another reverse genetics Sendai virus variant, RGV19, containing the six F mutations and neither of the M mutations of F1-R.  2-way and 3-way ligations with the pKS/SeV-19 and pRS3Gg constructs from previous studies are being used to generate RVG19 cDNA.  pKS/Sev-19 is a 13.7kb vector consisting of the F gene with the six mutations and the M gene with neither of the mutations.  pRS3Gg is a 19.2kb vector containing the entire Sendai virus genome.  Confirmation of a successful pRGV19 construct will be shown using appropriate restriction digest analysis.  The pRGV19 construct will then be used to make the reverse genetics virus, RGV19, in order to examine the effects of the mutations on enhanced cleavability of the F protein, tissue tropism, and pathogenicity in mice.  




1.            Ishida, N., and Homma, M.  1978.  Sendai Virus.  Adv. Virus Research.  23:  349-383.


2.            Okada, H., Seto, J.T., McQueen, N.L., Klenk, H.-D., Rott, R., and Tashiro, M.  1998.  Determinants of Pantropism of the F1-R Mutant of Sendai Virus:  Specific Mutations Involved are in the F and M Genes.  Arch. Virology.  143:  243-252.


3.            Tashiro, M., McQueen, N.L., Seto, J.T., Klenk, H.-D., and Rott, R.  1996.  Involvement of the Mutated M Protein in Altered Budding Polarity of a Pantropic Mutant, F1-R, of Sendai Virus.  Journal of Virology.  70(9):  5990-5997.


4.            Tashiro, M., Seto, J.T., Choosakul, S., Hegemann, H., Klenk, H.-D., and Rott, R.  1992.  Changes in Specific Cleavability of the Sendai Virus Fusion Protein:  Implications for Pathogenicity in Mice.  Journal of Gen. Virology.  73:  1575-1579. 


5.            Tashiro, M., Yamakawa, M., Tobita, K., Klenk, H.-D., Rott, R., and Seto, J.T.  1990.  Organ Tropism of Sendai Virus in Mice:  Proteolytic Activation of the Fusion Glycoprotein in Mouse Organs and Budding Site at the Bronchial Epithelium.  Journal of Virology.  64(8):  3627-3634.


6.            Tashiro, M., Yamakawa, M., Tobita, K., Seto, J.T., Klenk, H.-D., and Rott, R.  1990.  Altered Budding Site of a Pantropic Mutant of Sendai Virus, F1-R, in Polarized Epithelial Cells.  Journal of Virology.  64(10):  4672-4677.


7.            Tashiro, M., Yokogoshi, K., Tobita, K., Seto, J.T., Rott, R., and Kido, H.  1992.  Tryptase Clara, an Activating Protease for Sendai Virus in Rat Lungs, is Involved in Pneumopathogenicity.  Journal of Virology.  66:  7211-7216