Transactivation of Cellular Genes Involved in Nucleotide Metabolism by the Regulatory IE1 Protein of Murine Cytomegalovirus Is Not Critical for Viral Replicative Fitness in Quiescent Cells and Host Tissues

Abstract

Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-ΔIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-ΔIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-ΔIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs

“…As expected from our previous study (10), the ⌬ie1 mutant was found to be attenuated in comparison to the wt MCMV during acute infection of newborn mice. The cause of this attenuation was not the focus of this investigation, but our data and the observations of another group, who found that the doubling time for the titers of the ⌬ie1 mutant was significantly delayed in several organs of irradiated mice, including the lungs (62), are consistent with our previous suggestion (10) that the ⌬ie1 mutant displays increased sensitivity to the innate defense mechanisms of the host.…”

“…In contrast to the HCMV mutant, an MCMV ⌬ie1 deletion mutant grew with kinetics comparable to those of wild-type (wt) MCMV in various cell types in vitro, independently of whether infection was performed at a low or a high MOI (10). In vivo, the ⌬ie1 mutant displayed an attenuated phenotype (10); the doubling time of the viral titers was significantly reduced in various organs, including the lungs (62).…”

The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation

“…The in vivo replicative potential of viruses was determined by establishing virus growth curves for host tissues in the absence of immune control (61,70). Specifically, BALB/cJ mice were immunocompromised and infected (see above) with the viruses under investigation.…”

Synergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues

“…Transcript IE1 specifies the 89/76 kDa IE1 protein (Keil et al, 1985(Keil et al, , 1987b, which is involved in the early disruption of nuclear domains 10 (Ghazal et al, 2005;Maul, 2008) (Gribaudo et al, 2000;Lembo et al, 2000) and as a co-transactivator of virus early (E)-phase genes (Messerle et al, 1992). Whilst IE1 is dispensable for virus reactivation from latency (Busche et al, 2009) as well as for the transactivation of cellular genes involved in nucleotide metabolism (Wilhelmi et al, 2008), apparently due to redundant virus regulation of these crucial functions, IE1 contributes to virus fitness in vivo (Ghazal et al, 2005). Transcript IE3 specifies the 88-90 kDa IE3 protein, which is the essential transactivator of virus E-phase genes (Angulo et al, 2000;Messerle et al, 1992).…”

“…Interestingly, although the bipartite enhancer accelerates MIE gene expression through synergistic action of its two components, this kick-start advantage does not result in faster virus replication in cell culture or in host tissues. confidence intervals of DTs (given in parentheses) were calculated as described previously (Simon et al, 2006;Wilhelmi et al, 2008). Virus fitness was assessed by the DTs of (i) virus genome numbers, as determined by gB/M55-specific qPCR; (ii) infectivity, measured as p.f.u.…”