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Yellow fever virus proteins NS2A, NS2B, and NS4B: identification and partial N-terminal amino acid sequence analysis
T J Chambers1, D W McCourt, C M Rice
1Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
Abstract:
A series of fusion proteins corresponding to the hydrophobic ns2 and ns4 regions of yellow fever virus (YF) were generated in Escherichia coli using trpE fusion vectors. Antisera to ns2 and ns4 region fusion proteins recognize virus-specific proteins of 15 and 27 kDa, respectively. N-terminal amino acid sequence analysis of the 27-kDa protein indicates that the N-terminus of YF NS4B immediately follows a signalase-like cleavage site. Additional sequence data generated by microsequence analysis of labeled proteins immunoprecipitated with mouse hyperimmune antisera have identified the 15-kDa protein as NS2B and an additional 20-kDa viral protein as NS2A. Comparison of the sequences adjacent to the N-termini of these viral proteins suggests that three distinct types of cleavage events are involved in processing the hydrophobic YF ns2 and ns4 regions. These include cleavage after a short side chain amino acid to generate the N-terminus of NS2A, cleavage after two arginine residues to produce the N-terminus of NS2B, and a cleavage site consistent with the specificity of signalase to generate the N-terminus of NS4B. Analysis of virus-specific protein patterns in several different mammalian cell lines and in Aedes albopictus cells suggests that the same cleavage sites are used in different hosts. These findings are discussed in relation to the processing of flavivirus polyproteins.
Insights
Researchers identified distinct cleavage sites for processing yellow fever virus (YF) non-structural proteins NS2A, NS2B, and NS4B. These cleavage events occur consistently across different host cells, aiding flavivirus polyprotein processing understanding.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Yellow fever virus (YF) polyprotein processing is crucial for viral replication.
- The hydrophobic non-structural (NS) protein regions (NS2, NS4) are key targets for understanding viral maturation.
Purpose of the Study:
- To characterize the processing of hydrophobic YF virus non-structural proteins.
- To identify the specific cleavage sites involved in generating mature viral proteins.
Main Methods:
- Generation of trpE fusion proteins in Escherichia coli.
- Antisera production and Western blot analysis.
- N-terminal amino acid sequencing and microsequence analysis of labeled proteins.
Main Results:
- Fusion proteins identified virus-specific proteins of 15 kDa (NS2B) and 27 kDa (NS4B).
- N-terminal sequencing revealed signalase-like cleavage for NS4B, cleavage after arginine for NS2B, and cleavage after a short side chain for NS2A.
- Identified a 20-kDa viral protein as NS2A.
- Consistent cleavage site usage observed across mammalian and insect cell lines.
Conclusions:
- Three distinct cleavage mechanisms are involved in processing YF NS2 and NS4 regions.
- These cleavage sites are conserved across different host systems.
- Findings contribute to understanding flavivirus polyprotein processing and viral pathogenesis.