Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

Abstract

The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.Human cytomegalovirus (HCMV) is the largest and most complex member of the family of human herpesviruses. The virion of HCMV consists of three distinct structures, a nucleocapsid containing a double-stranded linear DNA genome, an envelope including an as-yet-undefined number of viral glycoproteins, and a tegument layer located between the capsid and the envelope (27,40,44,45). HCMV assembly is a multistage and poorly understood process. Although all proposed models include well-studied mechanisms of capsid assembly within the nucleus of infected cells, final tegumentation and envelopment in the cytoplasm of infected cells remain poorly understood (25). The assembly pathway and protein interactions that are required for formation of the tegument layer are not defined. As an example, the tegument protein ppUL69 is expressed only in the nucleus, whereas some tegument proteins such as pp150 (ppUL32) and pp28 (ppUL99) are expressed only in the cytoplasm during the replication of HCMV (33). Other tegument proteins such as ppUL53 and pp65 (ppUL83) are expressed in the nucleus of cells early in infection but are localized predominantly in the cytoplasm late in infection (33). Thus, it is unclear whether tegument proteins associate with the capsid in the nucleus or in the cytoplasmic assembly compartment at a later step (25,33). Electron microscopic studies have revealed that both nuclear and c...

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).…”