Cell cycle dependent replication of the murine cytomegalovirus
Creators: name: Muller, Mark T.
Publisher: name: University of British Columbia
Year: 2010

description: The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences.