scholarly journals Human Cytomegalovirus Virions Differentially Incorporate Viral and Host Cell RNA during the Assembly Process

2000 ◽  
Vol 74 (19) ◽  
pp. 9078-9082 ◽  
Author(s):  
Astrid E. Greijer ◽  
Chantal A. J. Dekkers ◽  
Jaap M. Middeldorp
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Rna ◽  
Gene Products ◽  
Active Infection ◽  
Virion Assembly ◽  
Viral Genes ◽  
Infected Cells ◽  
Low Levels ◽  
Cellular Dna ◽  
Positive Results

ABSTRACT While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

scholarly journals Human Cytomegalovirus Inhibits Cellular DNA Synthesis and Arrests Productively Infected Cells in Late G1

Virology ◽  
1996 ◽  
Vol 224 (1) ◽  
pp. 150-160 ◽  
Author(s):  
Wade A. Bresnahan ◽  
Istvan Boldogh ◽  
E.Aubrey Thompson ◽  
Thomas Albrecht
Keyword(s):  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Infected Cells ◽  
Cellular Dna

scholarly journals Retrieval of Human Cytomegalovirus Glycoprotein B from Cell Surface Is Not Required for Virus Envelopment in Astrocytoma Cells

2002 ◽  
Vol 76 (10) ◽  
pp. 5147-5155 ◽  
Author(s):  
Michael A. Jarvis ◽  
Kenneth N. Fish ◽  
Cecilia Söderberg-Naucler ◽  
Daniel N. Streblow ◽  
Heather L. Meyers ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Critical Role ◽  
Dominant Negative ◽  
Glycoprotein B ◽  
Virion Assembly ◽  
Virion Envelope ◽  
Infected Cells ◽  
Dynamin I ◽  
Golgi Network

ABSTRACT Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the trans-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.

scholarly journals Human Cytomegalovirus Infection Leads to Accumulation of Geminin and Inhibition of the Licensing of Cellular DNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2369-2376 ◽  
Author(s):  
Nilima Biswas ◽  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Dna Replication ◽  
Host Cell ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Early Protein ◽  
Infected Cells ◽  
Mcm Proteins ◽  
Cellular Dna

ABSTRACT Previous studies have shown that infection of G0-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.

scholarly journals Antisense Transcription in the Human Cytomegalovirus Transcriptome

2007 ◽  
Vol 81 (20) ◽  
pp. 11267-11281 ◽  
Author(s):  
Guojuan Zhang ◽  
Bindu Raghavan ◽  
Mark Kotur ◽  
Jacquelyn Cheatham ◽  
Daniel Sedmak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Antisense Transcription ◽  
Cdna Libraries ◽  
Viral Gene ◽  
Human Populations ◽  
Cdna Clones ◽  
Gene Products ◽  
Antisense Transcripts ◽  
Reading Frame ◽  
Viral Genes

ABSTRACT Human cytomegalovirus (HCMV) infections are prevalent in human populations and can cause serious diseases, especially in those with compromised or immature immune systems. The HCMV genome of 230 kb is among the largest of the herpesvirus genomes. Although the entire sequence of the laboratory-adapted AD169 strain of HCMV has been available for 18 years, the precise number of viral genes is still in question. We undertook an analysis of the HCMV transcriptome as an approach to enumerate and analyze the gene products of HCMV. Transcripts of HCMV-infected fibroblasts were isolated at different times after infection and used to generate cDNA libraries representing different temporal classes of viral genes. cDNA clones harboring viral sequences were selected and subjected to sequence analysis. Of the 604 clones analyzed, 45% were derived from genomic regions predicted to be noncoding. Additionally, at least 55% of the cDNA clones in this study were completely or partially antisense to known or predicted HCMV genes. The remarkable accumulation of antisense transcripts during infection suggests that currently available genomic maps based on open-reading-frame and other in silico analyses may drastically underestimate the true complexity of viral gene products. These findings also raise the possibility that aspects of both the HCMV life cycle and genome organization are influenced by antisense transcription. Correspondingly, virus-derived noncoding and antisense transcripts may shed light on HCMV pathogenesis and may represent a new class of targets for antiviral therapies.

scholarly journals Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

2020 ◽  
Vol 8 (12) ◽  
pp. 1928
Author(s):  
Sami Salmikangas ◽  
Jutta E. Laiho ◽  
Kerttu Kalander ◽  
Mira Laajala ◽  
Anni Honkimaa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Viral Rna ◽  
Quantitative Real Time Pcr ◽  
Virion Assembly ◽  
Rna Molecules ◽  
Branched Dna ◽  
Infected Cells ◽  
Good Signal

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.

scholarly journals Human herpesvirus 7 U47 gene products are glycoproteins expressed in virions and associate with glycoprotein H

2006 ◽  
Vol 87 (3) ◽  
pp. 501-508 ◽  
Author(s):  
Tomohiko Sadaoka ◽  
Koichi Yamanishi ◽  
Yasuko Mori
Keyword(s):  
Monoclonal Antibody ◽  
Virus Infection ◽  
Human Cytomegalovirus ◽  
Human Herpesvirus ◽  
Gene Products ◽  
Similar Function ◽  
Human Herpesvirus 6 ◽  
Glycoprotein H ◽  
Genes Encoding ◽  
Infected Cells

The function of the human herpesvirus 7 (HHV-7) U47 gene, which is a positional homologue of the genes encoding glycoprotein O (gO) in human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), was analysed. A monoclonal antibody (mAb) against the U47 gene product reacted in immunoblots with proteins migrating at 49 and 51 kDa in lysates of HHV-7-infected cells and with 49 and 51 kDa proteins in partially purified virions. Digestion of the 49 and 51 kDa proteins with endoglycosidase H and peptide N-glycosidase F indicated that the U47-encoded proteins were modified with N-linked oligosaccharides. Therefore, the U47 gene and its product were named gO, as in HCMV and HHV-6. In addition, the anti-gO mAb co-immunoprecipitated glycoprotein H (gH) in HHV-7-infected cells, indicating an association between HHV-7 gO and gH. The results suggest that the HHV-7 gO–gH complex might have a similar function to that in HCMV or HHV-6, such as cell–cell fusion in virus infection.

scholarly journals Human cytomegalovirus UL97 kinase alters the accumulation of CDK1

2012 ◽  
Vol 93 (8) ◽  
pp. 1743-1755 ◽  
Author(s):  
Rachel B. Gill ◽  
Scott H. James ◽  
Mark N. Prichard
Keyword(s):  
Human Cytomegalovirus ◽  
Kinase Activity ◽  
Cell Cycle Checkpoint ◽  
M Cell ◽  
Serine Threonine Kinase ◽  
Virion Assembly ◽  
Threonine Kinase ◽  
Nuclear Reorganization ◽  
Cycle Checkpoint ◽  
Infected Cells

The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G2/M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.

scholarly journals Human Cytomegalovirus Specifically Controls the Levels of the Endoplasmic Reticulum Chaperone BiP/GRP78, Which Is Required for Virion Assembly

2007 ◽  
Vol 82 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Nicholas J. Buchkovich ◽  
Tobi G. Maguire ◽  
Yongjun Yu ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Viral Protein Synthesis ◽  
Virion Assembly ◽  
Infected Cells ◽  
Virion Formation

ABSTRACT The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.

scholarly journals Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection

Open Biology ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 160298 ◽  
Author(s):  
Jonathan Pavelin ◽  
Dominique McCormick ◽  
Stephen Chiweshe ◽  
Saranya Ramachandran ◽  
Yao-Tang Lin ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Virion Assembly ◽  
Complex Process ◽  
Novel Host ◽  
Infected Cells ◽  
Human Cytomegalovirus Infection ◽  
Infection Inhibition ◽  
Or Gene ◽  
Assembly Compartment

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.

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