Sendai virion transcriptase complex: polyeptide composition and inhibition by virion envelope proteins.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Sendai virus is a paramyxovirus with an RNA genome.
• Viral RNA-dependent RNA polymerase (RdRp) is crucial for viral replication.
• Understanding the composition and regulation of viral polymerases is key to antiviral strategies.

Purpose of the Study:

• To isolate and characterize the RNA-dependent transcriptase activity associated with Sendai virus nucleocapsids.
• To identify the viral protein components responsible for transcriptase activity.
• To investigate the regulatory roles of viral envelope proteins and exogenous RNA on polymerase function.

Main Methods:

• Sendai virions were disrupted using Triton X-100 and KCl.
• Nucleocapsids and envelope proteins were separated by centrifugation.
• Buoyant density of nucleocapsids was determined using D2O sucrose gradients.
• RNA-dependent transcriptase activity was assayed and specific activities were compared.
• Polypeptide composition of active nucleocapsids was analyzed by molecular weight.
• Inhibitory effects of envelope proteins and stimulatory effects of yeast RNA were tested.

Main Results:

• Nucleocapsids, comprising 46% of virion proteins, exhibited a buoyant density of 1.29 gm/cm³.
• Nucleocapsid-associated transcriptase activity showed a ninefold higher specific activity than in disrupted virions.
• Active nucleocapsids contained two polypeptides: MW 75,000 and MW 60,000.
• Sendai virus envelope proteins (glycoproteins and matrix protein) inhibited polymerase activity.
• Heat denaturation abolished the inhibitory effect of envelope proteins.
• Yeast RNA stimulated the nucleocapsid-associated enzyme activity.

Conclusions:

• Sendai virus RNA-dependent transcriptase activity is primarily associated with nucleocapsids.
• The viral polymerase complex involves specific polypeptides (MW 75,000 and 60,000).
• Viral envelope proteins act as negative regulators of polymerase activity, suggesting a role in viral assembly or release.
• Polyanions like yeast RNA may stimulate polymerase activity by interacting with the enzyme-template complex.