Related Experiment Videos
Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene
1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
Abstract:
With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.
Insights
Researchers identified a viral glycoprotein encoded by the A33R gene in vaccinia virus. This protein is crucial for virus spread and is found on the outer envelope of extracellular enveloped virions.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Vaccinia virus, a member of the Orthopoxvirus genus, possesses a complex envelope structure.
- Understanding the proteins involved in viral envelope formation is key to comprehending virus dissemination.
Purpose of the Study:
- To identify the gene encoding a specific glycoprotein on the vaccinia virus outer envelope.
- To characterize the properties and localization of this viral glycoprotein.
- To investigate the conservation and potential role of this protein in orthopoxviruses.
Main Methods:
- Utilized three monoclonal antibodies (MAbs) to detect and characterize a viral glycoprotein.
- Employed techniques including Western blotting (under reducing and nonreducing conditions), DNA transfection, gene sequencing, and Triton X-114 partitioning.
- Performed electron microscopy to determine protein localization on viral particles.
Main Results:
- A glycoprotein, encoded by the A33R gene, was identified on the outer envelope of vaccinia virus.
- This protein is conserved across multiple orthopoxviruses and is a type II integral membrane protein with glycosylation sites.
- The A33R gene product is specifically present on extracellular enveloped virions, not intracellular mature virions.
Conclusions:
- The A33R gene product is a conserved viral glycoprotein essential for vaccinia virus dissemination.
- Its localization on extracellular enveloped virions suggests a role in virus entry or spread.
- Further research into this protein could inform strategies against orthopoxvirus infections.