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.Cytomegalovirus (CMV), a betaherpesvirus, is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. It may, however, cause severe disease in the absence of an effective immune response, as in immunologically immature and immunocompromised individuals (41). Strict species specificity has hampered investigation of human CMV (HCMV) in its natural host, and infection of mice with murine CMV (MCMV) has been extensively used as a model for studying the pathogenesis of HCMV infection (40,52,63). The two viruses are biologically similar in replication and pathogenesis (34,50,51), and their homologous genomes display similar genetic organizations and encode analogous gene products with similar functions (56). However, the functions of many viral gene products remain to be explored in order to determine the interactions of CMV with the host cell and its pathogenic mechanisms.CMV replicates most efficiently in the absence of cellular DNA synthesis (23, 39). Moreover, it has evolved mechanisms to inhibit progression through the cell cycle and arrests cells with a G 1 DNA content (5,18,47,53,60,68). The viral DNA polymerase thus has competition-free access to the DNA precursors, and CMV replicates efficiently in vitro in quiescent cells (18,45) and infects terminally differentiated cells in vivo (41, 51). However, since postmitotic cells do not replicate their genomes, the very low levels of deoxyribonucleotides (dNTP) and cell functions involved in DNA synthesis limit viral replication.It has been d...

Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene

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“…Cells infected with MCMV mutants that lack any of these genes undergo apoptosis. Moreover, these mutants have been shown to be attenuated in vitro (5,6,29) and in vivo (11,25).…”

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A Human Protein Complex Homologous to the Drosophila MSL Complex Is Responsible for the Majority of Histone H4 Acetylation at Lysine 16

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