The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis

Abstract

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.

FIG. 1.

Identification of the MCMV M45 protein by the specific antiserum. (A) Immunoblot analysis of native or recombinant M45 expression in MCMV-infected NIH 3T3 cells, in cells transfected with an M45 expression vector, or in E. coli. Proteins of whole-cell lysates were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a membrane, and probed with the M45 antiserum. Lanes: 1, extracts from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h after infection with MCMV; 3, extracts from NIH 3T3 cells transiently transfected with the control vector pcDNA3; 4, extracts from NIH 3T3 cells transiently transfected with the M45 expression vector pcDNA3-M45; 5, extracts from IPTG-induced E. coli containing the M45 expression vector pET30-M45. (B) Immunoprecipitation of M45 by the specific antiserum. M45 was immunoprecipitated from cell extracts of MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by immunoblotting with the M45 antiserum. Lanes: 1, cell extract from infected cells; 2, cell extract immunoprecipitated with the M45 antiserum; 3, cell extract immunoprecipitated with preimmune serum. Sizes of the molecular mass markers are shown on the left of each panel. Asterisk indicates the Ig heavy chains recognized by the secondary antibody.

FIG. 2.

(A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.

FIG. 3.

Detection of M45 in purified MCMV particles by immunoblotting. Proteins from a whole-cell lysate of MCMV-infected NIH 3T3 cells at 48 hpi (lane 1) or from virus particles purified by two rounds of centrifugation through a 15% sucrose cushion (lane 2) or via density gradient centrifugation (lane 3) were separated by SDS-PAGE, blotted onto a membrane, and probed with antibodies against the viral proteins M45 and M44 and the cellular proteins R2 and actin.

FIG. 4.

RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane 2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column. (B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 μg) before and after the addition of a constant amount of mouse R1 protein (2 μg). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 μg. (C) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 (10 μg) together with a constant amount of mouse R1 (2 μg) or of the catalytically inactive R1 C429A protein (9 μg). The increasing amounts of purified recombinant M45 are 0, 7, and 14 μg. (D) Allosteric inhibition of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1-R2 alone or together with recombinant M45 protein was assayed in the presence of 0, 20, 80, or 400 μM dATP.

FIG. 5.

Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected. Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1, anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP + UTP + ATP + GTP + dCTP + dTTP + dATP + dGTP) to minimize variations due to small differences in cell number in the samples.

FIG. 6.

(A) Survival curves of SCID mice infected with a wild-type virus (MCMV-GFP) or with an M45 mutant virus (IIIG2) expressing GFP. (B) Survival curves of SCID mice infected with wild-type (bd-MCMV), revertant (MCMV-REV), or M45 mutant (BamX) viruses without GFP. SCID mice were injected intraperitoneally with 10⁶ PFU of virus and were monitored daily for survival. (C) Accumulation of infectious viruses in the spleen, liver, kidneys, lungs, and salivary glands. Two SCID mice for each group were infected as described above. The animals were killed 24 days after infection, and total levels of virus in homogenates of target organs were determined by plaque assays on NIH 3T3 cells. Each data point is the average for two animals.

FIG. 7.

Line diagram representing the amino acid sequence alignment of M45 with the R1 proteins of E. coli, HSV-1, and EBV. The proposed nucleotide-binding site (GxGxxG) and the five cysteines and two tyrosines that have a direct catalytic role, as discussed in the text, are highly conserved in the E. coli, HSV-1, and EBV R1 proteins. By contrast, most of these residues are absent in the M45 sequence.