Transfer of the UAP56 Interaction Motif of Human Cytomegalovirus pUL69 to Its Murine Cytomegalovirus Homolog Converts the Protein into a Functional mRNA Export Factor That Can Substitute for pUL69 during Viral Infection

Abstract

Nucleocytoplasmic shuttling and interaction with the cellular mRNA export factor UAP56 are prerequisites for the mRNA export activity of human cytomegalovirus (HCMV) pUL69. Although the murine cytomegalovirus homolog pM69 shuttles, it fails to export mRNAs due to its inability to recruit UAP56. However, chimeric proteins comprising pM69 fused to N-terminal pUL69 fragments, including its UAP56 interaction motif, acquire mRNA export activity. Importantly, growth curves of recombinant HCMVs illustrate that such a chimeric protein, but not pM69, substitutes for pUL69 during HCMV infection.

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.…”

“…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).…”

“…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).…”

“…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).…”

“…The integrity of all newly generated plasmids was confirmed by automated DNA sequence analysis. The bacterial artificial chromosome (BAC) HB15, which contains the genomic sequence of the HCMV laboratory strain AD169 and its derivative HB15⌬pUL69, from which the genomic sequence of UL69 was removed, have been described before (9,37). To engineer recombinant HCMVs harboring amino acid substitutions within the UL69 gene, linear recombination cassettes were utilized to revert the HB15⌬pUL69 BACmid by a two-step recombination strategy (38).…”