Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

Abstract

Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

“…The failure of viral proteins to localize at the AC after inhibition of viral DNA synthesis by GCV, is in concert with previous studies using anti-HCMV molecules. 13,64 Interestingly, RhoB was still able to be recruited at the HCMV AC, even when the Late phase of the viral life-cycle was inhibited by GCV and proper AC was not formed (Fig. 5, h-i and q-r).…”

“…The endosomal sorting complex required for transport (ESCRT) machinery and multivesicular bodies (MVB) play a role in virion maturation. [6][7][8][9][10][11][12][13][14] RhoB belongs to the Rho GTPase family of proteins that comprises 6 subfamilies. 15 The RhoA subfamily consists of RhoA, RhoB and RhoC, which exhibit approximately 85% amino acid sequence identity.…”

Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells

“…Neither the UL133/8 locus nor UL135 and UL136 are required for VAC formation in fibroblasts, indicating unique membrane-trafficking requirements in ECs. The HCMV tegument proteins pUL48, pUL94, and pUL103 have been shown to be required for VAC formation in fibroblasts (74); however, it is not known if these proteins are required in ECs. Defining the mechanisms by which viral proteins function to mediate VAC formation will contribute to our understanding of cellular trafficking pathways in ECs.…”

Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

“…In cells infected with UL36-deleted HSV-1 and PRV, the newly assembled capsids accumulate in the cytoplasm (26,27) and this event appears to result from the failure of recruitment of the cytoplasmic capsid to the site of secondary envelopment (28). Recently, it was shown that in the absence of pUL48 expression, development of the cytoplasmic virion assembly complex (cVAC) was abrogated (29).…”