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Mutational analysis, using a full-length rubella virus cDNA clone, of rubella virus E1 transmembrane and cytoplasmic
1Department of Pathology and Laboratory Medicine, Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.
Abstract:
We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.
Insights
Researchers created a rubella virus clone to study E1 protein domains. Mutations in E1
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Rubella virus is a significant human pathogen.
- The E1 protein is crucial for rubella virus assembly and infectivity.
- Understanding E1's role in virus assembly is essential for developing antiviral strategies.
Purpose of the Study:
- To investigate the role of the E1 transmembrane and cytoplasmic domains in rubella virus assembly.
- To determine how specific mutations in these domains affect viral replication and release.
Main Methods:
- Construction of a full-length infectious rubella virus cDNA clone (pBRM33).
- Site-directed mutagenesis to introduce alanine substitutions in the E1 transmembrane and cytoplasmic domains.
- Analysis of viral RNA replication, protein synthesis, transport, and virus release.
Main Results:
- Mutations in E1 transmembrane and cytoplasmic domains did not significantly impact RNA replication or protein synthesis.
- Most mutations reduced virus release, with cysteine 467 and leucine 471 substitutions causing dramatic decreases in virus yield.
- E2/E1 heterodimer formation remained unaffected by the mutations.
Conclusions:
- The E1 transmembrane and cytoplasmic domains are critical for efficient rubella virus assembly and release.
- Specific residues within these domains play a key role in the late stages of virus budding.
- Findings support the hypothesis that E1 interacts with nucleocapsids to facilitate virus egress.