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Altered plaque formation by recombinant vaccinia virus expressing simian immunodeficiency virus Nef
1International Laboratory of Molecular Biology for Tropical Disease Agents, Department of Veterinary Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
Abstract:
The nef gene of primate lentiviruses encodes a myristoylated protein that is important for pathogenicity and the maintenance of high virus loads. A deletion in nef leads to a significant reduction of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. At the cellular and biochemical levels, Nef has been shown to down-regulate CD4 and major histocompatibility complex class I molecules and to interact with cellular protein kinases. The importance of these activities for Nef function remains uncertain. We have prepared vaccinia virus recombinants expressing different alleles of SIV nef. When grown on TK- 143 cells, recombinants constructed with the nef allele from SIVmac1A11 produced typical plaques while recombinants expressing the nef allele from SIVmac239-R1 gave rise to plaques with altered morphology. By using chimeric Nef proteins and site-directed mutagenesis, the amino acid responsible for altered plaque formation was mapped to a leucine at residue 211. In vitro phosphorylation of immunoprecipitates prepared from cells infected with the vaccinia virus recombinants resulted in labeled proteins of 62 and 90 kDa. The recombinants differed in the ability to stimulate phosphorylation, and the leucine at residue 211 was again found to be the determining amino acid. These results might help elucidate the role of nef in the pathogenesis of SIV.
Insights
The simian immunodeficiency virus (SIV) nef gene is crucial for pathogenicity. A specific leucine residue (L211) in Nef protein significantly impacts viral plaque formation and phosphorylation, offering insights into SIV pathogenesis.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The nef gene in primate lentiviruses encodes a myristoylated protein vital for pathogenicity and high viral loads.
- Deletions in nef reduce simian immunodeficiency virus (SIV) pathogenicity in macaques.
- Nef protein's roles in down-regulating CD4 and MHC class I, and interacting with kinases, are known but their functional importance is unclear.
Purpose of the Study:
- To investigate the functional significance of specific amino acids within the SIV Nef protein.
- To elucidate the role of Nef in SIV pathogenesis by analyzing its effects on viral replication and cellular processes.
Main Methods:
- Generation of vaccinia virus recombinants expressing different SIV nef alleles.
- Analysis of plaque morphology on TK-143 cells.
- Site-directed mutagenesis and chimeric protein construction to map functional domains.
- In vitro phosphorylation assays of immunoprecipitated Nef proteins.
Main Results:
- Recombinants with different SIV nef alleles exhibited distinct plaque morphologies.
- A leucine at residue 211 (L211) was identified as critical for altered plaque formation.
- L211 also determined the ability of Nef recombinants to stimulate in vitro phosphorylation of 62 and 90 kDa proteins.
Conclusions:
- The leucine at residue 211 in SIV Nef is a key determinant of altered plaque formation and phosphorylation activity.
- These findings contribute to understanding the role of nef in SIV pathogenesis and viral replication.