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.

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-DeltaIE1. 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-DeltaIE1, 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-DeltaIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.