scholarly journals Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome

2004 ◽  
Vol 78 (20) ◽  
pp. 10960-10966 ◽  
Author(s):  
Susan M. Varnum ◽  
Daniel N. Streblow ◽  
Matthew E. Monroe ◽  
Patricia Smith ◽  
Kenneth J. Auberry ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Proteins ◽  
Capillary Liquid Chromatography ◽  
Open Reading Frames ◽  
Cellular Proteins ◽  
Ion Cyclotron Resonance ◽  
Virion Protein ◽  
Enveloped Virus ◽  
Dense Bodies ◽  
Large Complex

ABSTRACT Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

scholarly journals Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus

2020 ◽  
Vol 8 (6) ◽  
pp. 820
Author(s):  
Yohann Couté ◽  
Alexandra Kraut ◽  
Christine Zimmermann ◽  
Nicole Büscher ◽  
Anne-Marie Hesse ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Kinase Activity ◽  
Current Knowledge ◽  
Protein Complexes ◽  
Cellular Proteins ◽  
Structural Proteins ◽  
Virion Protein ◽  
Host Proteins ◽  
Essential Prerequisite

The assembly of human cytomegalovirus (HCMV) virions is an orchestrated process that requires, as an essential prerequisite, the complex crosstalk between viral structural proteins. Currently, however, the mechanisms governing the successive steps in the constitution of virion protein complexes remain elusive. Protein phosphorylation is a key regulator determining the sequential changes in the conformation, binding, dynamics, and stability of proteins in the course of multiprotein assembly. In this review, we present a comprehensive map of the HCMV virion proteome, including a refined view on the virion phosphoproteome, based on previous publications supplemented by new results. Thus, a novel dataset of viral and cellular proteins contained in HCMV virions is generated, providing a basis for future analyses of individual phosphorylation steps and sites involved in the orchestrated assembly of HCMV virion-specific multiprotein complexes. Finally, we present the current knowledge on the activity of pUL97, the HCMV-encoded and virion-associated kinase, in phosphorylating viral and host proteins.

scholarly journals ORF018R, a highly abundant virion protein from Singapore grouper iridovirus, is involved in serine/threonine phosphorylation and virion assembly

2008 ◽  
Vol 89 (5) ◽  
pp. 1169-1178 ◽  
Author(s):  
Fan Wang ◽  
Xuezhi Bi ◽  
Li Ming Chen ◽  
Choy-Leong Hew
Keyword(s):  
Viral Proteins ◽  
Open Reading Frames ◽  
Serine Phosphorylation ◽  
Embryonic Cells ◽  
Virion Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Particle Assembly ◽  
Virion Assembly ◽  
Eukaryotic Translation ◽  
Late Genes

Iridovirus is an important pathogen causing serious diseases among wild, cultured and ornamental fish. Previous studies have shown that Singapore grouper iridovirus (SGIV) contains 162 open reading frames (ORFs) from which 51 viral proteins have been confirmed by proteomics studies. ORF018R, which is conserved among vertebrate iridoviruses, is an abundant virion protein identified from SGIV. Here, immunofluorescence staining showed that ORF018R occurred at high abundance throughout SGIV-infected cells. The function of ORF018R was explored using antisense morpholino oligonucleotides (asMOs). Knockdown of ORF018R expression resulted in a reduction in the expression of viral late genes, distortion of viral particle assembly and inhibition of SGIV infection in grouper embryonic cells. Western blotting with phosphoserine-specific antibody indicated that serine phosphorylation was significantly enhanced for proteins of molecular masss 17–32 kDa by SDS-PAGE when ORF018R expression was eliminated. These proteins were analysed further by two-dimensional gel electrophoresis, and numerous protein spots were found to shift to a lower pI and higher molecular mass as a result of the loss of ORF018R function. Five proteins with enhanced phosphorylation were identified by matrix-assisted laser desorption/ionization time-of-flight (TOF)-TOF mass spectrometry, including three viral proteins: ORF049L (dUTPase), ORF075R and ORF086R, and two host proteins: subunit 12 of eukaryotic translation factor 3 and natural killer enhancing factor. These findings suggest that ORF018R is involved in serine/threonine phosphorylation in SGIV-infected late-stage cells and plays an important role in expression of viral late genes and virion assembly.

The proteome of human cytomegalovirus virions and dense bodies is conserved across different strains

2015 ◽  
Vol 204 (3) ◽  
pp. 285-293 ◽  
Author(s):  
Nicole Büscher ◽  
Christina Paulus ◽  
Michael Nevels ◽  
Stefan Tenzer ◽  
Bodo Plachter
Keyword(s):  
Human Cytomegalovirus ◽  
Dense Bodies ◽  
Different Strains

scholarly journals The Human Cytomegalovirus UL74 Gene Encodes the Third Component of the Glycoprotein H-Glycoprotein L-Containing Envelope Complex

1998 ◽  
Vol 72 (10) ◽  
pp. 8191-8197 ◽  
Author(s):  
Mary T. Huber ◽  
Teresa Compton
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Protein Species ◽  
Gene Encoding ◽  
Peptide Backbone ◽  
Glycoprotein H ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391–5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090–3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.

scholarly journals Defective HIV-1 proviruses produce viral proteins

2020 ◽  
Vol 117 (7) ◽  
pp. 3704-3710 ◽  
Author(s):  
Hiromi Imamichi ◽  
Mindy Smith ◽  
Joseph W. Adelsberger ◽  
Taisuke Izumi ◽  
Francesca Scrimieri ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Viral Proteins ◽  
Open Reading Frames ◽  
Peripheral Blood Mononuclear ◽  
Hiv Rna ◽  
Rna Transcripts ◽  
Extracellular Virus ◽  
Hiv 1

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as “defective” by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant “graveyard” of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell–cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

scholarly journals Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Caroline Lehmann ◽  
Jessica Julia Falk ◽  
Nicole Büscher ◽  
Inessa Penner ◽  
Christine Zimmermann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antibody Response ◽  
Human Cytomegalovirus ◽  
Protein Complex ◽  
Vaccine Development ◽  
Laboratory Strain ◽  
Neutralizing Antibody ◽  
Critical Question ◽  
Dense Bodies ◽  
The Impact

ABSTRACTThe development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCEInfections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

scholarly journals The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

2012 ◽  
Vol 87 (3) ◽  
pp. 1720-1732 ◽  
Author(s):  
Eva Maria Borst ◽  
Jennifer Kleine-Albers ◽  
Ildar Gabaev ◽  
Marina Babić ◽  
Karen Wagner ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Unit Length ◽  
Viral Proteins ◽  
Enzymatic Process ◽  
Genome Packaging ◽  
Subnuclear Localization ◽  
Promising Target ◽  
Synthetic Ligand ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACTCleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

scholarly journals Global Proteomic Changes Induced by the Epstein-Barr Virus Oncoproteins Latent Membrane Protein 1 and 2A

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Robert M. DeKroon ◽  
Harsha P. Gunawardena ◽  
Rachel Edwards ◽  
Nancy Raab-Traub
Keyword(s):  
Membrane Protein ◽  
Epstein Barr Virus ◽  
Viral Proteins ◽  
Latent Membrane Protein ◽  
Cellular Proteins ◽  
Latent Membrane Protein 1 ◽  
Barr Virus ◽  
Mediated Effects ◽  
Proteasome Subunits ◽  
Epstein Barr

ABSTRACTThe Epstein-Barr virus (EBV) oncoproteins latent membrane protein 1 (LMP1) and LMP2A constitutively activate multiple signaling pathways, and both have been shown to interact with cellular ubiquitin ligases and affect cellular ubiquitination. To detect the LMP1- and LMP2A-mediated effects on the global cellular proteome, epithelial cell lines expressing LMP1 or LMP2A were analyzed using label-free quantitative proteomics. To identify proteins whose ubiquitination is affected by the viral proteins, the cells were cultured in the presence and absence of deubiquitinase (DUB) and proteasome inhibitors. More than 7,700 proteins were identified with high confidence and considerably more proteins showed significant differences in expression in the presence of inhibitors. Few of the differentially expressed proteins with or without inhibitors were common between LMP1 and LMP2A, confirming that the viral proteins induce unique changes in cell expression and function. However, ingenuity pathway analysis (IPA) of the data indicated that LMP1 and LMP2A modulate many of the same cellular regulatory pathways, including cell death and survival, cell movement, and actin filament dynamics. In addition, various proteasome subunits, ubiquitin-specific peptidases and conjugating enzymes, vesicle trafficking proteins, and NF-κB and mitogen-activated protein kinase signaling proteins were affected by LMP1 or LMP2A. These findings suggest that LMP1 and LMP2A may commonly target critical cell pathways through effects on distinct genes, with many cellular proteins modified by ubiquitination and/or degradation.IMPORTANCEThe Epstein-Barr virus proteins latent membrane protein 1 and 2 have potent effects on cell growth and signaling. Both proteins bind to specific ubiquitin ligases and likely modulate the cellular proteome through ubiquitin-mediated effects on stability and intracellular location. In this study, a comprehensive proteomic analysis of the effects of LMP1 and LMP2A revealed that both proteins affected proteasome subunits, ubiquitin-specific conjugases and peptidases, and vesical trafficking proteins. The data suggest that the effects of these proteins on the abundance and ubiquitination of cellular proteins are in part responsible for their effects on cell growth regulation.

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