Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in nuclear egress of capsids into cytoplasm, especially for the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remained unclear how ORF33 is incorporated into virions. In this study, we first showed that the endogenous ORF33 protein co-localizes with capsid proteins at discrete areas in the nucleus during viral infection. Co-sedimentation analysis as well as immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. Immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we showed that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 as a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.
IMPORTANCE:
Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between capsid and envelope in a herpesvirion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions both in the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we showed that ORF33 is associated with intranuclear capsids prior to primary envelopment and identified novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids an early stage of virion maturation process for herpesviruses.
