Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

Abstract

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for EpsteinBarr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.

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).…”

“…Through constructing and analyzing an ORF33 null mutant, we demonstrated that ORF33 is not required for viral genome replication or gene expression, but is essential for virion morphogenesis and egress. More interestingly, ORF33 null mutation caused a partial retention of nucleocapsids in the nucleus, and maturation of virions was arrested at a cytoplasmic stage of partially tegumented nucleocapsids, indicating that ORF33 participates in both primary envelopment of nucleocapsids and morphogenesis of virions in the cytoplasm (Guo et al, 2009). We also found that ORF33 localizes in the nucleus during early infection and interacts with another nuclear tegument protein (Guo et al, unpublished data).…”

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

“…In these viruses, ORF52 encodes a small protein of ϳ20 kDa. Most of the knowledge about gammaherpesvirus ORF52 has been obtained with 7,19,57). It appears that, MuHV-4 ORF52 is essential for tegumentation and secondary envelopment (6,57).…”

Role of RRV ORF52 in Virion Maturation

“…BAC into 293T cells, intracellular particles retrieved by freeze thaw of cells showed particles that lacked ORF33 and ORF45, but that had ORF52 at levels similar to wt BAC (120). In cells transfected with 33STOP BAC, particles were unable to undergo secondary envelopment and egress (120).…”

“…The majority of functional data regarding the herpesvirus tegument comes primarily from research on alpha and betaherpesviruses, which has shown that tegument proteins function in crucial roles in viral replication, including transcytosis of the herpesvirus capsid toward the nucleus during initial infection and egress from the nucleus toward the periphery during lytic replication (27,113,187,295,350). Additional functions of tegument proteins include modulation of the host cell environment during the immediate-early phase of infection (294), including shut off of host gene expression (289,301,302), antagonism of innate antiviral host response (102,147,178,180,276,362), and assembly and egress of herpesvirus virions ( (27,57,120,277) and reviewed in (213)). …”