Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus.
We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA(r)) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.
Kronenwett
Ralf
R
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, DKFZ-ATV, INF 242, 69120, Heidelberg, Germany.
Weisshart
Klaus
K
Knopf
Charles W
CW
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D020869:Gene Expression Profiling; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase