In this paper, we report that MHV-68 ORF52
encodes a capsid-associated tegument protein essential for tegumentation/envelopment of nucleocapsids in the cytoplasm and subsequent egress from infected cells. ORF52
is a highly expressed gene with true-late kinetics, activated after viral DNA replication (12
). Mutation of ORF52
in the MHV-68/BAC genome leads to arrest of the lytic phase of infection after viral genome replication, late gene expression, viral DNA cleavage/packaging, and nucleocapsid assembly in the nucleus but prior to complete virion tegumentation and envelopment in the cytoplasm and egress of infectious virions from the cell.
Partially tegumented virion particles isolated from cells transfected with the MHV-68 BAC ORF52
-null mutant clone (52S) morphologically appear similar to nucleocapsids and contain the four conserved capsid proteins required to form the icosahedral herpesvirus capsid, a structure MHV-68 shares with all other herpesviruses (6
). The 52S particles also contain at least one tegument protein found in extracellular virions, the conserved ORF64/UL36 homologue (25
). Interestingly, the HSV-1 UL36 protein has been shown to have deubiquitinating activity (24
); whether this function is conserved in the ORF64 homologue is not known. Another conserved primary tegument protein thought to be involved in egress of nucleocapsids into the cytoplasm, the ORF67/UL34 homologue (15
), was also found in association with 52S particles but not WT MHV-68 virions. ORF67 in MHV-68 infection may mimic UL34, which dissociates from capsids in the cytoplasm and is not present in mature, extracellular alphaherpesvirus virions (29
). If so, the loss of ORF52
may result in a defect in virion maturation prior to this step or inefficient dissociation of ORF67 from C capsids that have egressed into the cytoplasm. Moreover, 52S particles contain no ORF45, a protein less tightly bound to the nucleocapsid than ORF52 is. As such, 52S particles likely represent an arrested stage of virion morphogenesis, at which nascent nucleocapsids associated with ORF67 and ORF64 have egressed from the nucleus into the cytoplasm. Indeed, immature virions accumulate in the cytoplasm of cells transfected with 52S (Table ). ORF52 protein appears to be required to efficiently progress through the next stage of virion assembly, i.e., the loss of ORF67 from nascent nucleocapsids, association with outer tegument and glycoproteins required for canonical tegumentation and envelopment (wrapping), and egress from the cell. The existence of distinct, layered subdomains within the MHV-68 tegument has been recently suggested by electron cryotomography (W. Dai and Z. H. Zhou, submitted for publication). Consistent with this proposed function as a tegument protein, MHV-68 ORF52 localizes to the cytoplasm of transfected and infected cells. An EGFP-ORF52 fusion protein localized to the cytoplasm in distinct subcellular structures juxtaposed to the Golgi apparatus-derived network.
How exactly ORF52 functions is unclear. Understanding the mechanism of ORF52's function(s) requires a better understanding of the protein's interactions with other viral proteins and cellular machinery involved in the formation and release of virions from the infected cell. Studies have identified alphaherpesvirus tegument proteins that are specifically required for virion morphogenesis in the cytoplasm, such as pseudorabies virus UL37 (25
) and UL48 in both pseudorabies virus and HSV-1 (16
). Undoubtedly, other tegument proteins are also involved in these processes. For instance, conserved herpesvirus tegument proteins ORF67/UL34, ORF69/UL31, and ORF64/UL36 have been implicated in the egress of nucleocapsids from the nucleus and capsid transport in the cytoplasm (27
). In addition, a number of tegument proteins, including ORF52, are unique to each subfamily of herpesvirus and may have distinct roles in the formation and egress of virions. The complete set of tegument proteins encoded by each herpesvirus genome accomplishes a conserved evolutionary function, facilitating egress and entry of nucleocapsids through the cytoplasm. It has been recently suggested that herpesvirus nucleocapsids and tailed DNA bacteriophages may share a common ancestor (3
). In such a scenario, tegument proteins represent a subsequent adaptation to coping with another cellular layer, the cytoplasm, that had engulfed the common ancestor's original host, the primordial nucleus. Subsequent structural and genomic divergence has preserved this essential tegument function yet allowed incorporation of divergent proteins with effects on the infectivity of incoming virions, such as the gammaherpesvirus ORF45 (21
) and gammaherpesvirus UL41 virion host shutoff protein (34
). In this light, ORF52 protein has an apparently central role in tegumentation and egress and thus is an evolutionary link between the gammaherpesvirus proteome and the host cell.
In summary, we have analyzed the function of the ORF52 tegument protein in the MHV-68 model of the gammaherpesvirus lytic phase. The lytic phase of infection among gammaherpesviruses, such as, for example, KSHV, has been implicated in tumor establishment and progression, as well as reactivation and spread of the virus within patients, from tissues initially infected to B cells or lymphatic epithelial cells (8
). Understanding the production of virions is important for understanding the transmission and pathogenesis of these pathogens and developing therapeutic interventions in EBV- and KSHV-associated diseases.