Distinct Domains within the Human Cytomegalovirus UL26 Protein Are Important for Wildtype Viral Replication and Virion Stability

Abstract

The human cytomegalovirus (HCMV) UL26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.

Complex Expression of the UL136 Gene of Human Cytomegalovirus Results in Multiple Protein Isoforms with Unique Roles in Replication

“…Interestingly, the loss of latency phenotype was absent in UL136null myc virus infection, suggesting that other isoforms likely antagonize the suppressive action of the 23-and 19-kDa isoforms. The interplay of protein isoforms to control viral functions in an antagonistic fashion has also been shown in other herpesviruses (55,62). Further, we recently demonstrated an antagonistic relationship between two other UL133/8 proteins, where UL138 suppresses viral replication, while UL135 promotes viral replication in multiple cell types (27).…”

“…In both HCMV and Kaposi's sarcoma-associated herpesvirus (KSHV), ribosomal profiling has dramatically increased the estimates of viral coding capacity from both transcriptional and translational mechanisms (44,(46)(47)(48). Further, examples of multiple proteins originating from a single gene using multiple TISs in herpesviruses have recently been reported (27,(51)(52)(53)(54)(55), although in most cases the complexities of the transcription units were not evaluated. As most of these examples indicate that 2 to 3 TISs are encoded within a single ORF, the finding that UL136 encodes more than 6 is intriguing.…”

The Human Cytomegalovirus UL26 Protein Antagonizes NF-κB Activation

“…The wild-type (WT) HCMV strain used in this study was BADwt, a bacterial artificial chromosome (BAC) clone of Ad169 (26,27). The ⌬U L 26 mutant in these studies is a BADwt derivative that was previously described (15,28). Viral stocks were prepared through combining clarified infected cell medium (3,000 ϫ g) with the remaining clarified infected-cell lysate.…”

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators

“…Most of these ISG15 binding and all of UBE1L binding in yeast assays were also detected by co-immunoprecipitation (co-IP) assays (S4 Fig and S2 Table). Among them, the UL26 gene encodes the tegument proteins, p27 and p21, which are produced using two in-frame start codons and are shown to regulate viral gene expression, NF-κB signaling, and virion stability [71][72][73][74]. Since UL26 interacted with both ISG15 and UBE1L and its role in viral growth was relatively well reported compared to others, we further investigated the interaction of pUL26 with the ISG15 pathway.…”