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Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins
Published on: January 17, 2014
Syncytium formation by recombinant vaccinia viruses carrying bovine parainfluenza 3 virus envelope protein genes
Abstract:
The highly syncytium-inducing M strain and the weakly syncytium-inducing SC strain of bovine parainfluenza 3 virus differ by a single amino acid substitution in each of the hemagglutinin-neuraminidase (HN) and membrane (M) proteins, while their fusion (F) proteins are identical (T. Shioda, S. Wakao, S. Suzu, and H. Shibuta, Virology 162:388-396, 1988). We constructed recombinant vaccinia viruses which express separately the M virus HN (Vac-MHN), SC virus HN (Vac-SCHN), M virus M (Vac-MM), SC virus M (Vac-SCM), and common F (Vac-F) proteins. CV-1 cells were infected with the recombinants, singly or in combination, and implanted onto indicator MDBK cells for syncytium formation. Combinations of Vac-MHN plus Vac-F and Vac-SCHN plus Vac-F induced extensive and weak syncytium formation, respectively. Vac-F alone did not induce syncytium formation, and both Vac-MM and Vac-SCM had no effect on syncytium formation. These findings indicated that the syncytium formation by bovine parainfluenza 3 virus requires both the F and HN proteins and that the extensive syncytium formation by the M virus is due to the M virus HN protein. MSC, another weakly syncytium-inducing virus variant, newly isolated from the M virus, was identical to the M virus in the primary structure of the HN and M proteins but differed from the M virus by a single amino acid residue in the F protein. The combination of the recombinant vaccinia virus expressing the MSC virus F protein and Vac-MHN resulted in weak syncytium formation.
Insights
Bovine parainfluenza 3 virus syncytium formation requires both fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The M strain
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Bovine parainfluenza 3 virus (BPIV3) causes respiratory disease in cattle.
- The M and SC strains of BPIV3 exhibit different syncytium-inducing abilities.
- Differences in the hemagglutinin-neuraminidase (HN) and membrane (M) proteins, but not the fusion (F) protein, distinguish the M and SC strains.
Purpose of the Study:
- To elucidate the roles of BPIV3 HN, M, and F proteins in syncytium formation.
- To determine which viral protein contributes to the differential syncytium-inducing properties of BPIV3 strains.
Main Methods:
- Construction of recombinant vaccinia viruses expressing individual BPIV3 proteins (HN, M, F) from M and SC strains.
- Infection of CV-1 cells with recombinant viruses and co-culture with indicator MDBK cells.
- Assessment of syncytium formation to evaluate the function of each viral protein.
Main Results:
- Co-expression of the M strain HN (Vac-MHN) and F (Vac-F) proteins induced extensive syncytia.
- Co-expression of the SC strain HN (Vac-SCHN) and F (Vac-F) proteins induced weak syncytia.
- Neither M nor SC strain M proteins (Vac-MM, Vac-SCM) alone or with Vac-F induced syncytia, indicating HN and F are essential.
- A newly isolated variant, MSC, with an F protein mutation, showed weak syncytium formation with Vac-MHN.
Conclusions:
- Both BPIV3 F and HN proteins are necessary for syncytium formation.
- The HN protein of the M strain is responsible for extensive syncytium formation.
- Specific amino acid substitutions in the F protein can modulate syncytium-inducing activity.
