An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

Abstract

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

Role of tegument proteins in herpesvirus assembly and egress

“…In HCMV, the pUL11 homolog pUL99 has been found to interact with the pUL16 homolog pUL94 (Liu et al, 2009). Consistent with the previously described model of capsid transport and budding, herpesviruses lacking pUL16, pUL21 or pUL11 (or their homologs) have defects in virus egress, resulting in decreased amounts of extracellular viruses produced and the accumulation of non-enveloped capsids in the cytoplasm (MacLean et al, 1989(MacLean et al, , 1992Baines and Roizman, 1992;Baines et al, 1994;Kopp et al, 2003;Schimmer and Neubauer, 2003;Silva et al, 2003;Britt et al, 2004;Jones and Lee, 2004;Silva et al, 2005;Seo and Britt, 2006;Guo et al, 2009).…”

Tegument Proteins of Human Cytomegalovirus

“…In HCMV-infected cells, pp28 colocalizes with other tegument and viral glycoproteins at jux-tanuclear structures likely derived from the Golgi apparatus that presumably represent the site of viral assembly and final envelopment (100,157). Both myristoylation and a cluster of acidic residues (amino acids 44 to 59) are required for this localization and for incorporation into virions (79,158,164).…”

“…However, viruses that express only the amino-terminal 61 (164) or 57 (79) amino acids replicate almost as well as wildtype virus, indicating that a significant region (ϳ75%) of the carboxy terminus of the protein is dispensable. Deletion from the C terminus to amino acid 50 produces viable viruses with significant growth defects (164,165), and deletions to amino acid 43 are not viable (79). Deletion of the stretch of acidic residues between amino acids 44 and 59 also leads to the inability to recover infectious virus.…”

Nuclear Export of the Human Cytomegalovirus Tegument Protein pp65 Requires Cyclin-Dependent Kinase Activity and the Crm1 Exporter

“…The immature virions are then transported to the final site of envelopment in the cytoplasm. The acquisition of the tegument proteins likely occurs in both the nucleus and the cytoplasm, since the steady-state localization of some HCMV tegument proteins is restricted during infection (1,7,22,26). The distribution of other tegument proteins is temporally regulated (5,11,18,27).…”