The US16 Gene of Human Cytomegalovirus Is Required for Efficient Viral Infection of Endothelial and Epithelial Cells

Bronzini, Matteo; Luganini, Anna; Dell’Oste, Valentina; Andrea, Marco De; Landolfo, Santo; Gribaudo, Giorgio

doi:10.1128/jvi.06310-11

Cited by 31 publications

(63 citation statements)

References 50 publications

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“…HCMV TR was derived from an ocular specimen (47), and after a few passages on fibroblasts, it was cloned into a bacterial artificial chromosome (48,49). Reconstitution of infectious TR was performed as previously described (50) by cotransfecting HFFs with the corresponding TR bacterial artificial chromosome and a plasmid expressing HCMV pp71. Reconstituted infectious virus retained the ability to infect endothelial and epithelial cells, as well as monocytes and macrophages (49,50).…”

Section: Hcmv Preparations and Infection Conditionsmentioning

confidence: 99%

“…Reconstitution of infectious TR was performed as previously described (50) by cotransfecting HFFs with the corresponding TR bacterial artificial chromosome and a plasmid expressing HCMV pp71. Reconstituted infectious virus retained the ability to infect endothelial and epithelial cells, as well as monocytes and macrophages (49,50). HCMV VR1814 is a derivative of a clinical isolate recovered from a cervical swab of a pregnant woman (51).…”

Section: Hcmv Preparations and Infection Conditionsmentioning

confidence: 99%

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Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Pignoloni

Fionda

Dell’Oste

et al. 2016

The Journal of Immunology

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Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I–related chain (MIC) A/B and UL16-binding proteins (ULBP)1–6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell–mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3–related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

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Section: Hcmv Preparations and Infection Conditionsmentioning

confidence: 99%

Section: Hcmv Preparations and Infection Conditionsmentioning

confidence: 99%

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Pignoloni

Fionda

Dell’Oste

et al. 2016

The Journal of Immunology

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“…Most known restrictions to HCMV tropism in ECs occur early in the infection process, residing at the level of viral entry (62) or delivery of the viral genome to the nucleus (58). As such, one plausible explanation for the observed defect in replication may be that a protein encoded in the UL133-UL138 locus is required for viral entry into ECs, similar to the UL128-UL131A locus.…”

Section: Ul133-ul138mentioning

confidence: 99%

“…Studies in MCMV revealed a requirement for the M45 gene to inhibit apoptosis for productive replication specifically in ECs and macrophages (57). Recently, the US16 HCMV gene was reported to be required for delivery of viral genomes and tegument proteins to the nuclei of ECs but not fibroblasts (58). Viruses containing disruptions in US16 are defective at the immediate-early (IE) stage and all subsequent stages of infection in ECs.…”

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confidence: 99%

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

Bughio

Elliott

Goodrum

2013

J Virol

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Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb= region of the HCMV genome as important for the latent infection in CD34؉ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138 NULL virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138 NULL virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138 NULL virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138 NULL virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138 NULL virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.

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“…Nonetheless, very little is known about the expression patterns and functions of individual US12 proteins in infected cells. In this regard, the intracellular localization of the US14, US16, US17, and US18 proteins was determined by immunofluorescence analyses that revealed an association with the cytoplasmic virion assembly compartment (cVAC), thus suggesting that their functions may be linked to virion maturation and egress (13,14). In support of this hypothesis, it was observed that inactivation of the US17 gene in producer fibroblasts results in increased production of noninfectious viral particles that can, in turn, deliver augmented amounts of the pp65 immunomodulatory tegument protein to newly infected cells, thus altering the regulation of both intrinsic and innate responses of cells infected with the US17-deficient virus (15).…”

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confidence: 99%

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

Cavaletto

Luganini

Gribaudo

2015

J Virol

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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...

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The US16 Gene of Human Cytomegalovirus Is Required for Efficient Viral Infection of Endothelial and Epithelial Cells

Cited by 31 publications

References 50 publications

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

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

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

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