Inactivation of the Human Cytomegalovirus
            <i>US20</i>
            Gene Hampers Productive Viral Replication in Endothelial Cells

Cavaletto, Noemi; Luganini, Anna; Gribaudo, Giorgio

doi:10.1128/jvi.01141-15

Cited by 20 publications

(25 citation statements)

References 48 publications

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“…US16, US20, and US21, members of the HCMV US12 family of putative seven-transmembrane domain proteins (66), were O-glycosylated at the C terminus (Fig. 1B), suggesting that their orientation in the membrane could be opposite to that of GPCRs (67). The secreted RANTES-specific chemokine receptor UL22A (also known as UL21.5) was found heavily glycosylated, as previously reported (68,69).…”

Section: Mapping O-glycosites In Human Herpesvirusessupporting

confidence: 49%

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

Bagdonaite

Nordén

Joshi

et al. 2016

Journal of Biological Chemistry

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Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a "bottom up" mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.

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Section: Mapping O-glycosites In Human Herpesvirusessupporting

confidence: 49%

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

Bagdonaite

Nordén

Joshi

et al. 2016

Journal of Biological Chemistry

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“…In fact, inactivation of some US12 gene members affects virusmediated manipulation of the host innate immune response (37,38) or, as we reported for US16 (12) and US20 (39), HCMV cell tropism. However, irrespective of the final outcome for virus-host cell interactions, the functions of several US12 proteins appear to be related to the late stages of HCMV maturation and the fine-tuning of the most appropriate protein composition of virions (38; this study).…”

Section: Fig 10mentioning

confidence: 82%

“…Infectious recombinant TRΔUS16, TRUS16stop, TRUS16HA-UL130V5, TRΔUS16-UL130V5, TRUS16HAΔC-UL130V5, TRwt, and Towne viruses were reconstituted in HFFs by transfection of the corresponding BAC DNA as previously described (12,39). Viral titers were determined by plaque assay in HFFs.…”

Section: Methodsmentioning

confidence: 99%

“…The TRUS16HA-UL130V5 BAC (Fig. 1) was generated by a two-step replacement strategy using the galK recombineering method, as previously described (12,39,42,43). Briefly, in the first step the galK-kan cassette was amplified out of the pGalK-Kan plasmid (a gift from Dong Yu) by PCR using the UL130_galK/ kan primer set (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…For immunoblotting, the protein extractions, determination of protein concentrations, and immunoblot analysis were performed as previously described (12,39). Immunostaining was carried out with a mouse MAb against V5 or with a rat anti-HA MAb conjugated to horseradish peroxidase (clone 3F10; Roche).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer

Luganini

Cavaletto

Raimondo

et al. 2017

J Virol

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The human cytomegalovirus (HCMV) US12 gene family encodes a group of predicted seven-transmembrane proteins whose functions have yet to be established. While inactivation of individual US12 members in laboratory strains of HCMV does not affect viral replication in fibroblasts, disruption of the US16 gene in the low-passage-number TR strain prevents viral growth in endothelial and epithelial cells. In these cells, the US16-null viruses fail to express immediate early (IE), early (E), and late (L) viral proteins due to a defect which occurs prior to IE gene expression. Here, we show that this defective phenotype is a direct consequence of deficiencies in the entry of US16-null viruses in these cell types due to an impact on the gH/gL/UL128/UL130/UL131A (pentamer) complex. Indeed, viral particles released from fibroblasts infected with US16-null viruses were defective for the pentamer, thus preventing entry during infections of endothelial and epithelial cells. A link between pUS16 and the pentamer was further supported by the colocalization of pUS16 and pentamer proteins within the cytoplasmic viral assembly compartment (cVAC) of infected fibroblasts. Deletion of the C-terminal tail of pUS16 reproduced the defective growth phenotype and alteration of virion composition as US16-null viruses. However, the pentamer assembly and trafficking to the cVAC were not affected by the lack of the C terminus of pUS16. Coimmunoprecipitation results then indicated that US16 interacts with pUL130 but not with the mature pentamer or gH/gL/gO. Together, these results suggest that pUS16 contributes to the tropism of HCMV by influencing the content of the pentamer into virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells. The virus infects a variety of cell types by exploiting different forms of the envelope glycoprotein gH/gL hetero-oligomers, which allow entry into many cell types through different pathways. For example, incorporation of the pentameric gH/gL/UL128/UL130/ UL131A complex into virions is a prerequisite for infection of endothelial and epithelial cells. Here, we show that the absence of US16, a thus far uncharacterized HCMV multitransmembrane protein, abrogates virus entry into endothelial and epithelial cells and that this defect is due to the lack of adequate amounts of the pentameric complex in extracellular viral particles. Our study suggests pUS16 as a novel viral regulatory protein important for shaping virion composition in a manner that influences HCMV cell tropism.

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A cluster of 3′ coterminal transcripts from US12–US17 locus of human cytomegalovirus

Ma²,

Liu³

et al. 2016

Virus Genes

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Among all the human cytomegalovirus (HCMV) gene families, US12 family is relatively undefined in their transcriptional profile and biological functions. In this study, the transcription pattern and characteristics of HCMV US12-US17 gene region were studied extensively. Twenty-three clones harboring US12 cDNA sequence were screened out from a late cDNA library of an HCMV clinical isolate, Han. Using a set of US12-US17 gene-specific probes, six transcripts from US12-US17 locus were detected by northern blot at late kinetics of the clinical isolate. One additional transcript was found in late RNA of HCMV strain AD169. No evidence showing these transcripts contain introns by reverse transcription PCR. 3' and 5' termini of these transcripts were confirmed by Rapid Amplification of cDNA Ends. A novel protein-coding region was predicted in the shorter US14 transcript with an alternative in-frame 5' translation initiation site compared to that of the previously predicted US14 ORF. Our findings demonstrate the existence of a cluster of 3' coterminal unspliced transcripts with distinct 5' transcriptional initiation sites originated from US12-US17 gene region in the late infection phase of an HCMV clinical strain.

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

Cited by 20 publications

References 48 publications

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer

A cluster of 3′ coterminal transcripts from US12–US17 locus of human cytomegalovirus

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