An Endothelial Cell-Specific Requirement for the
            <i>UL133-UL138</i>
            Locus of Human Cytomegalovirus for Efficient Virus Maturation

Self Cite

139

The mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb= region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34 ؉ hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation. IMPORTANCEMechanisms by which viruses persist in their host remain one of the most poorly understood phenomena in virology. Herpesviruses, including HCMV, persist in an incurable, latent state that has profound implications for immunocompromised individuals, including transplant patients. Further, the latent coexistence of HCMV may increase the risk of age-related pathologies, including vascular disease. The key to controlling or eradicating HCMV lies in understanding the molecular basis for latency. In this work, we describe the complex interplay between two viral proteins, pUL135 and pUL138, which antagonize one another in infection to promote viral replication or latency, respectively. We previously described the role of pUL138 in suppressing virus replication for latency. Here we demonstrate a role of pUL135 in overcoming pUL138-mediated suppression for viral reactivation. From this work, we propose that pUL135 and pUL138 constitute a molecular switch balancing states of latency and reactivation.

Section: Resultssupporting

confidence: 80%

“…6). We recently demonstrated that while the UL133/8 locus was required for virus maturation in endothelial cells, a virus lacking the entire UL133/8 locus, UL133/ 8 NULL , exhibited no defects for virus maturation in fibroblasts (42). However, the results reported here suggest that UL135 is required for maturation when UL138 is expressed.…”

Section: Discussioncontrasting

confidence: 54%

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Umashankar

Rak

Bughio

et al. 2014

Self Cite

139

“…So far, however, little is known about the functions exerted by viral tropism factors that act at a postentry stage and regulate, in a cell type-specific manner, subsequent stages of the virus replication cycle. In addition to the HCMV US20 and MCMV M45 (47) proteins, two other HCMV proteins were recently reported to contribute to HCMV tropism in endothelial cells by regulating post-immediate-early phases in the viral replication cycle (48,49,50). In these studies, the UL135 and UL136 proteins encoded within the UL133-to-UL138 locus of a clinical strain of the virus (48) were found to be required for efficient HCMV replication in primary endothelial cells (50).…”

Section: Discussionmentioning

confidence: 95%

Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

Cavaletto

Luganini

Gribaudo

2015

The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells. IMPORTANCE Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark ofHCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent a key target for viral dissemination and replication in the host, and to contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a postentry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis and help lead to the design of new antiviral strategies able to exploit these functions. Human cytomegalovirus (HCMV) is a ubiquitous opportunistic pathogen that persists throughout the lifetime of the infected host through both the chronic and latent states of infection. Generally, HCMV causes asymptomatic or mildly symptomatic infections in immunocompetent individuals. In contrast, prima...

“…In addition, in cells infected with a pUL71-deficient virus, viral proteins that normally associate with the cVAC are mislocalized (21). (vi) cVACs form in cells infected with highly passaged laboratory strains of HCMV that lack some genes present in wild-type viruses (4, 5, 8-15, 17, 20-23), as well as after infection with a low-passage-number virus that carries a nearly complete complement of HCMV genes (24). Interestingly, an HCMV locus spanning UL133 to UL138 is required for cVAC development in human microvascular endothelial cells, but not in embryonic lung fibroblasts (24), indicating that cell-specific viral factors can be involved.…”

mentioning

confidence: 99%

“…(vi) cVACs form in cells infected with highly passaged laboratory strains of HCMV that lack some genes present in wild-type viruses (4, 5, 8-15, 17, 20-23), as well as after infection with a low-passage-number virus that carries a nearly complete complement of HCMV genes (24). Interestingly, an HCMV locus spanning UL133 to UL138 is required for cVAC development in human microvascular endothelial cells, but not in embryonic lung fibroblasts (24), indicating that cell-specific viral factors can be involved. (vii) In addition to protein regulators of cVAC biogenesis, the HCMV microRNAs (miRNAs) miR UL112-1, US5-1, and US5-2 target mRNAs of several host proteins involved in regulation of the cellular secretory apparatus (25).…”

mentioning

confidence: 99%

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

Das

Ortiz

Gurczynski

et al. 2014

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.