scholarly journals The ORF7b Protein of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Is Expressed in Virus-Infected Cells and Incorporated into SARS-CoV Particles

2006 ◽  
Vol 81 (2) ◽  
pp. 718-731 ◽  
Author(s):  
Scott R. Schaecher ◽  
Jason M. Mackenzie ◽  
Andrew Pekosz
Keyword(s):  
Stop Codon ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Accessory Protein ◽  
Coding Region ◽  
Reading Frame ◽  
Golgi Compartment ◽  
Infected Cells ◽  
Viral Accessory Protein

ABSTRACT Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two—open reading frame 3a (ORF3a) and ORF7a—have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a −1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.

scholarly journals Expression of novel genes encoded by the paramyxovirus J virus

2008 ◽  
Vol 89 (6) ◽  
pp. 1434-1441 ◽  
Author(s):  
Philippa J. M. Jack ◽  
Danielle E. Anderson ◽  
Katharine N. Bossart ◽  
Glenn A. Marsh ◽  
Meng Yu ◽  
...  
Keyword(s):  
G Protein ◽  
Transcription Initiation ◽  
Genome Structure ◽  
Integral Membrane Protein ◽  
The Novel ◽  
Coding Region ◽  
Attachment Protein ◽  
Reading Frame ◽  
C Terminus ◽  
Infected Cells

Characterization of the J virus or, in keeping with recent nomenclature recommendations, J paramyxovirus (JPV) genome revealed a unique genome structure, consisting of eight genes in the order 3′-N-P/V/C-M-F-SH-TM-G-L-5′. The small hydrophobic (SH) protein and the transmembrane (TM) protein genes are predicted to encode proteins 69 and 258 aa in size, respectively. The 4401 nt attachment (G) protein gene, much larger than most other paramyxovirus attachment protein genes sequenced to date, encodes a putative 709 aa attachment protein and contains distally a second open reading frame (ORF-X) 2115 nt long. Experiments undertaken in this study were intended to confirm the sequence-based gene allocation of JPV and to determine if proteins encoded by the SH gene, the novel TM gene and ORF-X are expressed. Northern blot analyses carried out on mRNA purified from JPV-infected cells indicated that the putative transcription initiation and termination sequences flanking the SH and TM genes are functional, consistent with their allocation as discrete genes, although a high level of read-through was observed across almost all transcriptional boundaries. Probes specific to the G protein coding region and ORF-X both identified an mRNA species corresponding to the predicted length of the G gene, confirming sequence-based predictions. While the SH and TM proteins were both detected in infected cells, no evidence was found for the expression of ORF-X. Preliminary studies indicate that the novel TM protein is a type II glycosylated integral membrane protein, orientated with its C terminus exposed at the cell surface.

scholarly journals Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

2002 ◽  
Vol 76 (3) ◽  
pp. 1450-1460 ◽  
Author(s):  
S. Spaderna ◽  
H. Blessing ◽  
E. Bogner ◽  
W. Britt ◽  
M. Mach
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Component ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Protein Product ◽  
Clinical Isolates ◽  
Coding Region ◽  
Reading Frame ◽  
Infected Cells ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

scholarly journals Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

2009 ◽  
Vol 54 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Olivier Delelis ◽  
Sylvain Thierry ◽  
Frédéric Subra ◽  
Françoise Simon ◽  
Isabelle Malet ◽  
...  
Keyword(s):  
Viral Genome ◽  
Effective Concentration ◽  
Coding Region ◽  
Delayed Emergence ◽  
Infected Cells ◽  
High Level ◽  
Emergence Of Resistance ◽  
Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

scholarly journals Characterization of a Novel vpu-Harboring Simian Immunodeficiency Virus from a Dent's Mona Monkey (Cercopithecus mona denti)

2005 ◽  
Vol 79 (13) ◽  
pp. 8560-8571 ◽  
Author(s):  
Marie-Christine Dazza ◽  
Michel Ekwalanga ◽  
Monique Nende ◽  
Karhemere Bin Shamamba ◽  
Pitchou Bitshi ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Genomic Sequence ◽  
Democratic Republic Of Congo ◽  
Coding Region ◽  
Reading Frame ◽  
Immunodeficiency Virus ◽  
Primate Lentiviruses ◽  
Infected Cells ◽  
Mona Monkey ◽  
Hiv 1

ABSTRACT We report the identification of a new simian immunodeficiency virus (SIV), designated SIVden, in a naturally infected Dent's Mona monkey (Cercopithecus mona denti), which was kept as pet in Kinshasa, capital of the Democratic Republic of Congo. SIVden is genetically distinct from the previously characterized primate lentiviruses. Analysis of the full-length genomic sequence revealed the presence of a vpu open reading frame. This gene is also found in the virus lineage of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus (SIVcpz) and was recently described in viruses isolated from Cercopithecus nictitans, Cercopithecus mona, and Cercopithecus cephus. The SIVden vpu coding region is shorter than the HIV-1/SIVcpz and the SIVgsn, SIVmon, and SIVmus counterparts. Unlike Pan troglodytes schweinfurthii viruses (SIVcpzPts) and Cercopithecus monkey viruses (SIVgsn, SIVmon, and SIVmus), the SIVden Vpu contains the characteristic DSGXES motif which was shown to be involved in Vpu-mediated CD4 and IκBα proteolysis in HIV-1 infected cells. Although it harbors a vpu gene, SIVden is phylogenetically closer to SIVdeb isolated from De Brazza's monkeys (Cercopithecus neglectus), which lacks a vpu gene, than to Cercopithecus monkey viruses, which harbor a vpu sequence.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

scholarly journals Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

2005 ◽  
Vol 280 (16) ◽  
pp. 15518-15525 ◽  
Author(s):  
Diane E. Handy ◽  
Yufeng Zhang ◽  
Joseph Loscalzo
Keyword(s):  
Glutathione Peroxidase ◽  
Untranslated Region ◽  
Stop Codon ◽  
Vascular Dysfunction ◽  
Lipid Peroxides ◽  
Mrna Levels ◽  
Coding Region ◽  
Sv40 Promoter ◽  
Secis Element

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

scholarly journals The Agnogene of the Human Polyomavirus BK Is Expressed

1998 ◽  
Vol 72 (7) ◽  
pp. 6233-6236 ◽  
Author(s):  
Christine Hanssen Rinaldo ◽  
Terje Traavik ◽  
Allan Hey
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Bk Virus ◽  
Human Polyomavirus ◽  
Coding Region ◽  
Reading Frame ◽  
Host Cell Proteins ◽  
Infectious Cycle ◽  
Perinuclear Area

ABSTRACT Primate polyomavirus genomes all contain an open reading frame at the 5′ end of the late coding region called the agnogene. A simian virus 40 agnoprotein with unknown functions has previously been demonstrated. We now show that a BK virus agnoprotein appears in the perinuclear area and cytoplasm late in the infectious cycle. It is phosphorylated in vivo and coimmunoprecipitates with a subset of host cell proteins.

scholarly journals Variable In Vivo Hepatitis D Virus (HDV) RNA Editing Rates According to the HDV Genotype

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1572
Author(s):  
Samira Dziri ◽  
Christophe Rodriguez ◽  
Athenaïs Gerber ◽  
Ségolène Brichler ◽  
Chakib Alloui ◽  
...  
Keyword(s):  
Rna Editing ◽  
Stop Codon ◽  
Genome Replication ◽  
Hepatitis D ◽  
Reading Frame ◽  
Delta Antigen ◽  
B Virus ◽  
Virion Morphogenesis ◽  
Kinetics Of

Human hepatitis delta virus (HDV) is a small defective RNA satellite virus that requires hepatitis B virus (HBV) envelope proteins to form its own virions. The HDV genome possesses a single coding open reading frame (ORF), located on a replicative intermediate, the antigenome, encoding the small (s) and the large (L) isoforms of the delta antigen (s-HDAg and L-HDAg). The latter is produced following an editing process, changing the amber/stop codon on the s-HDAg-ORF into a tryptophan codon, allowing L-HDAg synthesis by the addition of 19 (or 20) C-terminal amino acids. The two delta proteins play different roles in the viral cell cycle: s-HDAg activates genome replication, while L-HDAg blocks replication and favors virion morphogenesis and propagation. L-HDAg has also been involved in HDV pathogenicity. Understanding the kinetics of viral editing rates in vivo is key to unravel the biology of the virus and understand its spread and natural history. We developed and validated a new assay based on next-generation sequencing and aimed at quantifying HDV RNA editing in plasma. We analyzed plasma samples from 219 patients infected with different HDV genotypes and showed that HDV editing capacity strongly depends on the genotype of the strain.

scholarly journals Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7

1999 ◽  
Vol 67 (5) ◽  
pp. 2225-2232 ◽  
Author(s):  
Gregory Govoni ◽  
François Canonne-Hergaux ◽  
Cheryl G. Pfeifer ◽  
Sandra L. Marcus ◽  
Scott D. Mills ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Functional Expression ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Raw264.7 Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Vitro Assay

ABSTRACT Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defectiveS. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

scholarly journals Expression and Characterization of a Novel Structural Protein of Human Cytomegalovirus, pUL25

1999 ◽  
Vol 73 (5) ◽  
pp. 3800-3809 ◽  
Author(s):  
Maria Concetta Battista ◽  
Giovanna Bergamini ◽  
Maria Cristina Boccuni ◽  
Fabio Campanini ◽  
Alessandro Ripalti ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Transcriptional Analysis ◽  
Structural Protein ◽  
Native Form ◽  
Flag Epitope ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.

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