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Comparative Study
. 2000 Nov;74(22):10729-36.
doi: 10.1128/jvi.74.22.10729-10736.2000.

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity

M Wagner  1 , D Michel, P Schaarschmidt, B Vaida, S Jonjic, M Messerle, T Mertens, U Koszinowski
Affiliations
Comparative Study

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity

M Wagner et al. J Virol. 2000 Nov.

Abstract

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

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Figures

FIG. 1
FIG. 1
Structural analysis of recombinant MCMV genomes. Recombinant MCMV genomes were compared with wt MCMV and revertant rMCMV genomes by restriction enzyme analysis and agarose gel electrophoresis (A) and Southern analysis (B). Specific fragments expected after cleavage with NotI and SfiI are listed in panel D and marked in panels A and B. (C) Genome structure of the virus constructs within the HindIII-G fragment that contains the M97 ORF. The probe used for Southern blotting is shown at the top. The genome structures of the different viruses and the locations of the NotI and SfiI restriction sites in the M97 gene region are depicted below.
FIG. 2
FIG. 2
Kinase assays with cytoplasmic and nuclear extracts of MCMV-infected cells. MEF were infected with the indicated viruses. Cell fractionation was performed at 36 h p.i., and cytoplasmic (A) and nuclear matrix fractions (B) were prepared and treated for protein kinase activity. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography. Differences in the phosphorylation patterns are indicated by arrows. (C) Parallel samples were used for Western blotting with a pUL97-specific antiserum. The putative pM97 phosphoprotein is marked by the white arrowhead.
FIG. 3
FIG. 3
Protein kinase assays with cytoplasmic and nuclear extracts of rVV-infected cells. CV1 cells were infected with the indicated vaccinia viruses and harvested 24 h p.i. For protein kinase assays, cell fractionation was performed. (A) Kinase assays were done with extracts from cytoplasm and nuclear matrix in parallel. For Western blotting, aliquots of these assays were separated by SDS–10% PAGE. Both the tagged M97 protein (B) and pUL97 using a polyclonal antiserum (C) were visualized by chemiluminescence. pM97 is indicated by white arrows, and pUL97 is marked by black arrows. cop, vaccinia virus strain Copenhagen; M97, rVV-M97 carrying the M97 ORF; UL97, rVV-UL97 carrying the UL97 ORF.
FIG. 4
FIG. 4
Indirect immunofluorescence of CV1 cells infected with vaccinia viruses. Cells were infected and indirect immunofluorescence was performed at 24 h p.i. (A and B) A primary antibody directed against the StrepTactinII tag and a secondary antibody conjugated with Texas red were used. Cells were infected with (A) vaccinia virus strain Copenhagen or (B) rVV-M97. (C) Cells were infected with rVV-UL97. pUL97 was visualized with a polyclonal UL97-specific antiserum and a secondary antibody conjugated with FITC, and cells were counterstained with Evans blue.
FIG. 5
FIG. 5
GCV phosphorylation in MCMV- and rVV-infected cells. The phosphorylation of GCV in MEF and 143B (tk−) cells after mock infection or infection with the indicated viruses was determined at 36 h p.i. for MCMV or 24 h p.i. for rVV. Mean values from five independent experiments ± standard error are shown. VV cop, Copenhagen strain.
FIG. 6
FIG. 6
Reduced virus release in cells infected with ΔM97-MCMV and UL97-MCMV. MEF were infected with the indicated viruses at an MOI of 1 (A) or 0.01 (B). Supernatants were harvested daily from days 0 to 4 p.i. Virus titers were determined by standard plaque assay.
FIG. 7
FIG. 7
Virus titers in salivary glands and lungs of infected mice. BALB/c mice (five animals per group) were injected i.p. with 1,000 PFU of the indicated viruses. One day p.i., mice were depleted of CD4+ and CD8+ T lymphocytes. Virus titers in organs were determined at 7 days p.i. using a standard plaque assay. The significance of the titers was determined as follows: salivary glands, significant (P < 0.005 for all titers compared); lungs, wt MCMV versus ΔM97-MCMV significant (P < 0.005); wt MCMV versus UL97-MCMV and UL97-MCMV versus ΔM97-MCMV not significant. DL, detection limit.
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References

    1. Brune W, Messerle M, Koszinowski U H. Forward with BACs: new tools for herpesvirus genomics. Trends Genet. 2000;16:254–259. - PubMed
    1. Chee M S, Lawrence G L, Barrell B G. Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase. J Gen Virol. 1989;70:1151–1160. - PubMed
    1. Cobbold S P, Jayasuriya A, Nash A, Prospero T D, Waldman H. Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature. 1984;312:548–550. - PubMed
    1. Coulter L J, Moss H W, Lang J, McGeoch D J. A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted. J Gen Virol. 1999;74:387–395. - PubMed
    1. Crnkovic-Mertens I, Messerle M, Milotic I, Szepan U, Kucic N, Krmpotic A, Jonjic S, Koszinowski U. Virus attenuation after deletion of the cytomegalovirus Fc receptor is not due to antibody control. J Virol. 1998;72:1377–1382. - PMC - PubMed

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