Elimination of ie1 Significantly Attenuates Murine Cytomegalovirus Virulence but Does Not Alter Replicative Capacity in Cell Culture

Similar to other herpesviruses, the transcription of the cytomegalovirus (CMV) genome during the lytic infection is temporarily regulated (for a review, see reference 47). The immediate-early (IE or α) genes are the first ones to be expressed in the replicative cycle, and their expression does not depend on prior viral protein synthesis. Together with some virion proteins, the IE products activate viral genes and alter the infected cell to generate an appropriate milieu that favors viral replication. Transcription of early (E or β) genes requires the expression of at least one of the IE proteins, and only after viral replication has started, the transcription of late (L or γ) genes proceeds. The majority of the CMV IE transcripts originate from the major IE (MIE) locus. This locus is structurally similar between human CMV (HCMV) and the closely related mouse CMV (MCMV) (14, 53, 59). The primary transcript from the MIE region is under the control of the strong MIE enhancer-promoter and is differentially spliced to generate two predominant transcripts, the ie1 transcript that consists of exons 1 to 4, and the ie2 transcript that is composed of exons 1 to 3 and 5. In HCMV, the ie1 and ie2 transcripts are translated into the acidic 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins, respectively (for a review, see reference 59). The corresponding IE transcripts of MCMV encode the acidic 89-kDa IE1 phosphoprotein and the 88-kDa IE3 protein (33, 34, 45). The MIE proteins of HCMV display multiple functions (for reviews, see references 19 and 47). Mostly based on data from transient transfection assays and in vitro analysis, these viral products have been shown to be promiscuous regulatory proteins. In particular, HCMV IE2 is capable of down-regulating transcription from its own promoter by binding to the cis repression signals and exhibits strong transactivating properties on HCMV early promoters as well as on heterologous viral promoters. This gene product has been also shown to transactivate a number of host genes, such as the thymidine kinase gene or the dihydrofolate reductase gene. A number of components of the basal transcription machinery (i.e., TBP, TFIIB, TFIID) and cellular transcription factors (i.e., CREB, CBP, c-Jun) have been reported to directly interact with HCMV IE2 (11, 25, 31, 39, 57). HCMV IE1, the most abundant IE product, autostimulates the MIE enhancer-promoter (15, 59, 60, 62), plays an accessory role in the IE2-mediated activation of HCMV early and late genes (40, 62), and increases transcription of the long terminal repeat of human immunodeficiency virus (67). It can also transactivate a limited number of cellular promoters, including the ones corresponding to the DNA polymerase α (26), dihydrofolate reductase (65), and prointerleukin-1β (29). Interaction of HCMV IE1 with a number of cellular regulatory proteins (i.e., CTF-1, p107) has also been described previously (51). In addition to their regulatory activities, HCMV IE1 and IE2 are involved in perturbing a variety of other cellular processes, including cell cycle regulation (10, 69), apoptosis (71), and cell architecture. In this connection, IE1 has been shown to interact with chromatin and to cause the dispersion of the promyelocytic leukemia (PML) protein-associated nuclear bodies known as PML oncogenic domains, nuclear domain 10, or PML bodies (1, 2, 30, 35, 70). Though the precise role of these structures has not been defined, it has been hypothesized that they are part of a host cell repression system for viral infections. In recent years, advances in the methodology that permits the manipulation of the CMV genome, in particular the application of the bacterial artificial chromosome (BAC) technology has enormously facilitated the generation of CMV mutants (6, 46). This has made it possible to begin elucidating the biological significance of the MIE locus during the course of CMV infection. In agreement with the multifunctional nature assigned to the MIE proteins, it has been found that HCMVs with deletions in either the ie1 or the ie2 gene exhibit severe phenotypes in cultured cells. Marchini and coworkers (41) reported that a recombinant virus lacking the majority of the ie2 gene was blocked at the early phase of gene expression and did not produce infection progeny. A number of other HCMV with defects in the ie2 gene have been generated and essentially have corroborated the importance of IE2 in the control of viral gene expression (27, 56, 68). Deletion of the ie1 gene of HCMV causes a drastic growth defect under conditions of low multiplicity of infection of primary fibroblasts (24, 48). This growth deficiency appears to be the result of a broad reduction of delayed-early gene transcription in the absence of functional IE1 and can be circumvented at a high viral input (20). Due to the species specificity of HCMV, MCMV replication in mice has been extensively employed as a well-established model system for the study of different aspects of HCMV pathogenesis, latency, and reactivation. However, a limited number of studies have addressed MCMV IE activities. Similar to its HCMV IE2 homologue, MCMV IE3 represents the master switch that determines the transition from IE to E expression, and accordingly, MCMV ie3-deficient mutants are completely replication defective (3). The fact that the IE1 proteins of human and murine CMV exhibit a very similar global molecular structure, despite sharing little homology between their nucleic acid or amino acid sequences, has led to the assumption that they might have analogous functions during the course of the infection (33, 34, 60, 61). In this respect, MCMV IE1 has been reported to activate heterologous promoters (36) and to cooperate with IE3 protein in the activation of MCMV early gene promoters (45). In a recent study, Tang and Maul (63) have reported that MCMV IE1, as in the HCMV system, localizes to PML bodies and disperses them. Moreover, these studies reported on the ability of MCMV IE1 to bind to host cell repressors and proposed that the levels of IE1 expression might determine the number of repressed/activated viral genomes and, hence, the efficiency of a productive MCMV infection. An MCMV with a frameshift in exon 4 of the ie1 gene that leads to a truncated IE1 protein has been constructed (46) and was shown to have some growth impairment on cultured fibroblasts. However, the phenotype of this recombinant virus has not been further analyzed, and the presence of BAC sequences replacing genomic regions required for the in vivo infection has restricted its use in mice. Thus, the precise roles exerted by this protein during the MCMV life cycle still remain elusive. Moreover, the contribution that IE1 makes to in vivo MCMV growth and pathogenesis is unknown. In the present study, we have directly addressed the relevance of IE1 on the replicative strategies of MCMV in vitro and in the natural host. Using a parental full-length MCMV genome, we have created and characterized an MCMV devoid of exon 4 of the ie1 gene and, hence, unable to synthesize the IE1 protein. We found that this recombinant virus replicated as efficiently as the parental or revertant MCMV in different cell types in culture. In contrast, the ie1-deficient MCMV showed a significantly attenuated in vivo replication capacity and virulence. Thus, the MCMV IE1 protein is not essential but promotes efficient viral growth in mice.