Characterization of the Betaherpesviral pUL69 Protein Family Reveals Binding of the Cellular mRNA Export Factor UAP56 as a Prerequisite for Stimulation of Nuclear mRNA Export and for Efficient Viral Replication

Abstract

UL69 of human cytomegalovirus (HCMV) encodes a pleiotropic transactivator protein and has a counterpart in every member of the Herpesviridae family thus far sequenced. However, little is known about the conservation of the functions of the nuclear phosphoprotein pUL69 in the homologous proteins of other betaherpesviruses. Therefore, eukaryotic expression vectors were constructed for pC69 of chimpanzee cytomegalovirus, pRh69 of rhesus cytomegalovirus, pM69 of murine cytomegalovirus, pU42 of human herpesvirus 6, and pU42 of elephant endotheliotropic herpesvirus. Indirect immunofluorescence experiments showed that all pUL69 homologs expressed by these vectors were localized to the cell nucleus. Coimmunoprecipitation experiments identified homodimerization as a conserved feature of all homologs, whereas heterodimerization with pUL69 was restricted to its closer relatives. Further analyses demonstrated that pC69 and pRh69 were the only two homologs that functioned, like pUL69, as viral-mRNA export factors. As we had reported recently that nucleocytoplasmic shuttling and interaction with the cellular DExD/H-box helicases UAP56 and URH49 were prerequisites for the nuclear-mRNA export activity of pUL69, the homologs were characterized with regard to these properties. Heterokaryon assays demonstrated nucleocytoplasmic shuttling for all homologs, and coimmunoprecipitation and mRNA export assays revealed that the interaction of UAP56 and/or URH49 with pC69 or pRh69 was required for mRNA export activity. Moreover, characterization of HCMV recombinants harboring mutations within the N-terminal sequence of pUL69 revealed a strong replication defect of viruses expressing pUL69 variants that were deficient in UAP56 binding. In summary, homodimerization and nucleocytoplasmic shuttling activity were identified as conserved features of betaherpesviral pUL69 homologs. UAP56 binding was shown to represent a unique characteristic of members of the genus Cytomegalovirus that is required for efficient replication of HCMV.On the basis of biological properties, genome structure, and comparisons of primary amino acid sequences, the family Herpesviridae has been subdivided into three subfamilies, the Alpha-, Beta-and Gammaherpesvirinae (Fig. 1A) (40). Separation of these subfamilies is estimated to have proceeded approximately 180 to 200 million years ago (12), with development of genera within each subfamily occurring by more recent events (32). The subfamily Betaherpesvirinae contains four genera, Cytomegalovirus, Muromegalovirus, Roseolovirus, and Proboscivirus (Fig. 1A). The most intensively studied betaherpesviruses belong to the genus Cytomegalovirus and include human cytomegalovirus (HCMV; species, Human herpesvirus 5), rhesus cytomegalovirus (RhCMV; species, Macacine herpesvirus 3), and chimpanzee cytomegalovirus (CCMV; species, Panine herpesvirus 2). The genus Muromegalovirus comprises murine cytomegalovirus (MCMV; species, Murid herpesvirus 1) and rat cytomegalovirus (RCMV; species, Murid herpesvirus 2). Human herpesvirus 6 (HHV6...

pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

“…7D, lanes 8 to 11 and 13). The latter observation confirms our previous finding that the combinatorial change of arginines 22,23,25, and 26 to alanine changes pUL69 into a UAP56-binding-deficient mutant (6,8,9). Interestingly, however, the arginines at positions 28 and 29 were not required for UAP56 interaction, since a mutant harboring the respective substitutions was coprecipitated by Myc-UAP56 (Fig.…”

“…In accordance with previous results, expression of FLAG-pUL69 yielded a significant increase of CAT protein levels compared to the mock-transfected sample, thus confirming that pUL69 exports unspliced CAT reporter RNA (Fig. 7B, compare lanes 2 and 1) (6,8). Next, pUL69 mutants harboring arginine-to-alanine substitutions of putative methylation sites were tested.…”

“…1A). Strikingly, three of these sites can be detected within the arginine-rich R1 box, which has been demonstrated before to be required for UAP56 interaction and mRNA export activity (6,8,9). Additionally, one site is located within the RS box which is relevant for mRNA binding of pUL69 (7).…”

“…More recently, we extended these findings and showed that UAP56/URH49 recruitment is well conserved within the cytomegaloviral pUL69 homologs pCh69 of chimpanzee cytomegalovirus (CCMV) and pRh69 of rhesus cytomegalovirus (RhCMV). This activity correlated with stimulatory effects of the respective proteins on mRNA export analogously to pUL69 (8,9). The importance of UAP56/URH49 recruitment for HCMV multiplication was ultimately confirmed by the observation that recombinant cytomegaloviruses that had deletions or point mutations of the UAP56 interaction motif of UL69 (UL69⌬R1⌬RS and UL69mutUAP) exhibited a severe replication defect compared to wild-type virus (8).…”

“…Indirect immunofluorescence and coimmunoprecipitation (CoIP) analyses as well as the CAT mRNA export assay were performed using transiently transfected HEK293T or HeLa cells exactly as described in detail in previous publications (8,9,13,39). Yeast two-hybrid analysis was performed by transformation of the yeast strain Y153 with plasmids encoding GAL4-AD-and GAL4-BD-fused PRMT6 or pUL69 followed by growth selection and filter lift assays as described before (42,43).…”