Studies of the SV40 major capsid protein VP1: Sequence alterations in temperature-sensitive mutants and the expression and characterization of recombinant VP1 produced in Escherichia coli
Abstract
Temperature-sensitive (ts) assembly mutants of the simian virus 40 (SV40) have been mapped to the gene for the major capsid protein VP1 and classified into three complementation groups: tsB, tsC and tsBC. Dideoxy sequencing techniques performed on the cloned mutant DNA fragments revealed the amino acid substitutions which impart temperature-sensitivity to the protein. The inferred amino acid substitutions include: Gly40 $\to$ Glu and Pro252 $\to$ Leu (tsC260); Gln54 $\to$ Lys (tsB228); Pro58 $\to$ Arg (tsB218); Ala71 $\to$ Thr (tsB204, tsB211, tsB265); Ala71 $\to$ Val and Glu83 $\to$ Asp (tsB8); Gly163 $\to$ Ala (tsB221); Ala166 $\to$ Thr (tsB201); Ala195 $\to$ Val (tsB4); Leu244 $\to$ Ile (tsC240). The results of this analysis and the previously inferred substitutions determined for tsC219 and the tsBC class have been transposed onto the recently determined x-ray crystallographic structure of VP1 to gain insight into how these amino acid substitutions can affect the function of the SV40 major capsid protein. To further examine the structure/function relationships of VP1, protein expression systems which synthesize large quantities of two recombinant VP1 proteins have been identified. These expression systems contain the $\lambda$ phage P$\sb{\rm L}$ promoter and are induced by heat in cell lines which contain a temperature-sensitive cI (cI$\sp{857}$) repressor. One of the recombinant proteins, 9A-VP1, has 9 additional amino acid residues at the amino terminal end of the full VP1 protein; the other fusion protein, NS1A-VP1, contains the amino terminal region of the influenza virus NS1 protein fused to the amino terminus of the 9A-VP1 protein. The recombinant proteins were recovered from induced cells as inclusion bodies and isolated using urea or guanidine hydrochloride. The proteins were renatured using several dialysis protocols. The refolded recombinant proteins react with a monoclonal antibody specific for native VP1. The DNA binding properties of the recombinant VP1 proteins were investigated using several assays. Filter binding and native agarose gel electrophoresis analyses showed that the proteins can bind to both SV40 and pBR329 DNA. Using monoclonal antibodies specific for VP1, the DNA bound to the recombinant VP1 could be precipitated. Further immunological analysis suggested that the recombinant proteins do not selectively interact with any specific restriction fragment of SV40.
Degree
Ph.D.
Advisors
Bina, Purdue University.
Subject Area
Biochemistry|Molecular biology
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