Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.
Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.
Wadsworth Center, University at Albany School of Public Health, Albany, New York, 12201-2002, USA.
L A
LA
McCue
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Anders
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics*; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase