ABSTRACT
Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.
Subject(s)
Capsid Proteins/physiology , Cytomegalovirus/physiology , Phosphoproteins/physiology , Viral Proteins/physiology , Amino Acid Sequence , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cell Line , Cytomegalovirus/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Assembly/physiology , Virus Replication/genetics , Virus Replication/physiologyABSTRACT
The human cytomegalovirus tegument protein UL69 has been shown to be required for efficient viral replication at low multiplicities of infection. Several functions have been associated with UL69, including its ability to regulate cell cycle progression, translation, and the export of viral transcripts from the nucleus to the cytoplasm. However, it remains unclear which, if any, of these activities contribute to the phenotype observed with the UL69 deletion mutant. UL69 has been shown to interact with the cellular protein SPT6. The functional significance of this interaction has never been examined in the context of an infection. To address this, we generated UL69 mutant viruses that were unable to interact with SPT6 and determined what effect these mutations had on virus replication. Abolishing UL69's ability to interact with the SPT6 protein inhibited virus replication to levels indistinguishable from those observed following infection with the UL69 deletion mutant. Surprisingly, abolishing UL69's interaction with SPT6 also resulted in the impairment of UL69 shuttling activity. Finally, we demonstrate that inhibition of SPT6 expression by short hairpin RNA (shRNA) knockdown inhibits wild-type virus replication. Taken together, our results demonstrate that UL69's ability to interact with SPT6 plays a critical role in viral replication.
