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1.
Clin Microbiol Infect ; 22(12): 1002.e9-1002.e14, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27554204

ABSTRACT

Rhinoviruses (RVs) are frequently detected respiratory viruses that cause mild common cold symptoms, but may also lead to more severe respiratory tract infections. The large number of RV types, classified into species A, B and C, hampers clear insights into the epidemiology and clinical significance of each RV type. The aim of this study was to map the circulation of RV types in the Amsterdam area. RV-positive nasopharyngeal and oropharyngeal samples, collected from 2007 to 2012 in the Academic Medical Centre (Amsterdam, the Netherlands), were typed based on the sequence of the region coding for capsid proteins VP4 and VP2. RV-A, RV-B and RV-C were found in proportions of of 52.4% (334/637), 11.3% (72/637), and 36.2% (231/637), respectively. We detected 129 of the 167 currently classified types. RVs circulated throughout the entire year with a peak in the autumn and a decline in the summer. Some RV types were observed throughout the entire sampling period and others had a more seasonal pattern. Nine RV-A and four RV-B novel provisionally assigned types were identified. This study provides an insight into the molecular epidemiology of RVs in the Amsterdam area. The RVs circulating are diverse and include several provisionally new types.


Subject(s)
Capsid Proteins/genetics , Common Cold/epidemiology , Rhinovirus/genetics , Rhinovirus/isolation & purification , Common Cold/virology , Genotyping Techniques , Humans , Molecular Epidemiology , Nasopharynx/virology , Netherlands/epidemiology , RNA, Viral/isolation & purification , Rhinovirus/classification , Seasons , Sequence Analysis, DNA
2.
Clin Microbiol Infect ; 22(8): 736.e9-736.e15, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27265373

ABSTRACT

Rhinovirus (RV) is a frequent pathogen in young children, eliciting symptoms ranging from common colds to wheezing illnesses and lower respiratory tract infections. The recently identified RV-C seems to be associated with asthma exacerbations and more severe disease, but results vary. We studied the prevalence and severity of infection with RV in an unselected birth cohort. Children with respiratory symptoms entered the symptomatic arm of the cohort and were compared with asymptomatic children. Severity of wheezing and other respiratory symptoms was registered. Respiratory viruses were evaluated using throat and nasopharyngeal swabs on first presentation and after recovery (wheezing children). RV genotyping was performed on RV-PCR positive samples. RV was the most prevalent respiratory virus and was found in 58/140 symptomatic children (41%), 24/96 (25%) control children and 19/74 (26%) wheezing symptomatic children after recovery (p <0.05) and did not differ between wheezing and non-wheezing symptomatic children-respectively, 42% (38/90) and 40% (20/50). RV-A was the most commonly detected species (40/68, 59%), followed by RV-C (22/68, 32%) and RV-B (6/68, 9%). RV-B was more frequently detected in asymptomatic children (5/6, p <0.05). There was no significant difference in the frequency of RV species between wheezing and non-wheezing symptomatic children. Children with RV mono-infection had more severe symptoms, but no association between RV species and severity of disease was seen. In an unselected birth cohort from the Netherlands with mild respiratory disease RV was the most prevalent respiratory virus. RV(-C) infection was not associated with more severe disease or wheezing.


Subject(s)
Picornaviridae Infections/epidemiology , Picornaviridae Infections/virology , Rhinovirus , Bacterial Infections , Case-Control Studies , Child, Preschool , Cohort Studies , Coinfection , Female , Follow-Up Studies , Humans , Infant , Male , Netherlands/epidemiology , Picornaviridae Infections/diagnosis , Picornaviridae Infections/drug therapy , Prevalence , Rhinovirus/classification , Rhinovirus/genetics , Seasons , Severity of Illness Index
3.
J Gen Virol ; 96(Pt 3): 571-579, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25395595

ABSTRACT

Pleconaril is a capsid inhibitor used previously to treat enterovirus infections. A pleconaril-resistant echovirus 11 (E11) strain was identified before pleconaril treatment was given in an immunocompromised patient. The patient was also treated with intravenous Ig (IVIg) for a long period but remained unresponsive. The pleconaril-resistant strains could not be neutralized in vitro, confirming IVIg treatment failure. To identify the basis of pleconaril resistance, genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M and I188L) in the pleconaril-resistant viruses were found in the pocket region of VP1. Modelling suggested that V119M could confer resistance, most probably due to the protruding sulfate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterized by a different substitution (I183M), resistance was suggested to also result from a similar mechanism, i.e. due to a protruding sulfate side chain of methionine. Our results showed that resistant strains that arise in vivo display different markers from those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIg treatment failure, we predict that one of these factors could be immune related. Thus, both IVIg and capsid inhibitors target the viral capsid and can induce mutations that can be cross-reactive, enabling escape from both IVIg and the drug. This could limit treatment options and should be investigated further.


Subject(s)
Antigens, Viral/metabolism , Antiviral Agents/pharmacology , Drug Resistance, Viral , Enterovirus B, Human/genetics , Enterovirus B, Human/immunology , Oxadiazoles/pharmacology , Antigens, Viral/genetics , Antiviral Agents/therapeutic use , Echovirus Infections/virology , Gene Expression Regulation, Viral/physiology , Humans , Immunoglobulins, Intravenous , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxadiazoles/therapeutic use , Oxazoles
4.
Clin Microbiol Infect ; 20(10): O640-7, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24438366

ABSTRACT

Human parechoviruses (HPeV) cause symptoms ranging from severe neonatal infections to mild gastrointestinal and respiratory disease. Use of PCR and genotyping has markedly improved the detection rate of HPeV but has simultaneously raised questions about the clinical relevance of positive tests. This retrospective study correlates positive HPeV1 or HPeV3 PCR tests in stools from children with their symptoms to determine clinical relevance. Children with HPeV1- or HPeV3-positive stool samples, as detected by real time RT-PCR and direct genotyping, between 2004 and 2008 were selected. Clinical data were retrospectively collected from the patient's files and results were compared. One hundred and thirty-eight children with positive HPeV1 (n = 112) or HPeV3 (n = 26) stool samples were identified. Significantly more HPeV3-infected children were neonates or infants younger than 6 months of age. Meningitis or sepsis-like illnesses were diagnosed most frequently and were found in significantly younger children. Almost half of HPeV1-infected children had an underlying disease. Mild gastrointestinal disease was seen most frequently in these children. There was no clear correlation between viral load (Ct value) and severity of symptoms. In conclusion, HPeV3 detected by PCR in stool samples is associated with clinically relevant disease. For HPeV1, a positive stool sample is mainly associated with symptoms in children with underlying disease.


Subject(s)
Feces/virology , Parechovirus/classification , Parechovirus/isolation & purification , Picornaviridae Infections/virology , Female , Gastrointestinal Diseases/epidemiology , Gastrointestinal Diseases/virology , Genotype , Humans , Infant , Infant, Newborn , Male , Meningitis/epidemiology , Meningitis/virology , Parechovirus/genetics , Picornaviridae Infections/classification , Picornaviridae Infections/epidemiology , Picornaviridae Infections/pathology , Real-Time Polymerase Chain Reaction , Retrospective Studies , Sepsis/epidemiology , Sepsis/virology , Viral Load
5.
J Virol Methods ; 185(2): 228-33, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22796036

ABSTRACT

Human enteroviruses (EVs) are the leading cause of CNS-associated disease in childhood. Identification of the EV types that patients are infected with is essential for monitoring outbreaks, the emergence of new types or variants, epidemiological surveillance and contributes to patient management. Rapid and sensitive molecular detection methods are frequently used to detect EVs/HPeVs directly from CSF. This requires that sensitive EV typing methods from CSF material need to be developed. In the present study two nested PCR-based typing assays were evaluated. The performance of the EV-A and -B specific nested PCR protocol and the Codehop-based PCR protocol were analyzed with several TCID(50)-titrated EV-A to D strains and 22 EV positive CSF samples. The EV-A and -B protocol was found to be more sensitive than the Codehop protocol. The Codehop protocol showed a high degree of aspecific amplification products when run on a gel, and required additional gel purification. The detection limit of the two protocols varied between the types, ranging from 0.1TCID(50)/mL sample to 10(6)TCID(50)/mL sample. From the 22 EV positive CSF samples, 15 (68%) samples were typed using either protocol. All samples were characterized as members of species B (E30 (9), CAV9 (2), E6 (1), E11 (1), E21 (1), E25 (1)). Three samples (E30 (2) and E25 (1)) could only be typed using the EV-B protocol. In this study, selected EV strains could be typed using both assays at low virus concentrations, typically found in CSF. However, the EV-A and -B protocol was more sensitive than the Codehop protocol for primary typing of CSF samples.


Subject(s)
Capsid Proteins/analysis , Enterovirus A, Human/classification , Enterovirus B, Human/classification , Enterovirus Infections/cerebrospinal fluid , Polymerase Chain Reaction/methods , 5' Untranslated Regions , Capsid Proteins/genetics , Electrophoresis, Agar Gel , Enterovirus A, Human/genetics , Enterovirus A, Human/isolation & purification , Enterovirus B, Human/genetics , Enterovirus B, Human/isolation & purification , Enterovirus Infections/virology , Genotyping Techniques/methods , Humans , Phylogeny , RNA, Viral/analysis , RNA, Viral/genetics , Sensitivity and Specificity , Viral Load
6.
J Gen Virol ; 91(Pt 1): 145-54, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19759239

ABSTRACT

Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.


Subject(s)
Genetic Variation , Genome, Viral , Parechovirus/classification , Parechovirus/genetics , RNA, Viral/genetics , Sequence Analysis , Cluster Analysis , Genotype , Humans , Molecular Sequence Data , Netherlands , Parechovirus/isolation & purification , Phylogeny , Picornaviridae Infections/virology , Recombination, Genetic
7.
Clin Infect Dis ; 42(2): 204-10, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16355330

ABSTRACT

BACKGROUND: Human parechoviruses (HPeVs) are members of the family Picornaviridae and are classified into 3 known serotypes: HPeV1, HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections are most commonly associated with mild respiratory or gastrointestinal symptoms and occasionally with severe disease conditions, such as flaccid paralysis and encephalitis. HPeV3 infection has been associated with transient paralysis and neonatal infection and has until now only been reported in Japan and Canada. METHODS: Culture isolates considered to be enterovirus on the basis of cell culture but that were found to be enterovirus negative by 5' untranslated region reverse-transcriptase polymerase chain reaction (5'UTR RT-PCR) during the period December 2000 through January 2005 were selected. Isolates were tested by HPeV 5'UTR RT-PCR and were genotyped by sequencing the VP1 region. Phylogenetic analysis was performed, and the association with clinical symptoms was established. RESULTS: Thirty-seven (12%) of the 303 isolates that tested positive for enterovirus by cell culture were in fact HPeV. The majority of the HPeV-positive isolates (n = 27) could be identified as HPeV1. The remaining 10 isolates, which were grown from samples obtained in 2001, 2002, and 2004, could be typed as the recently identified HPeV3. HPeV was exclusively detected in children aged < 3 years. Children infected with HPeV3 were significantly younger than children infected with HPeV1, and sepsis-like illness and central nervous system involvement were more frequently reported in children infected with HPeV3. CONCLUSIONS: We report HPeV infections in young children during the period of 2000-2005 and show an association between HPeV3 infection and sepsis-like illness and central nervous system involvement in neonates.


Subject(s)
Parechovirus/classification , Parechovirus/isolation & purification , Picornaviridae Infections/epidemiology , Picornaviridae Infections/virology , Central Nervous System Infections/virology , Child, Preschool , Female , Gastrointestinal Diseases/virology , Genotype , Humans , Infant , Male , Netherlands/epidemiology , Parechovirus/genetics , Phylogeny , Respiratory Tract Infections/virology , Seasons
8.
Eur J Immunol ; 23(9): 2242-9, 1993 Sep.
Article in English | MEDLINE | ID: mdl-7690326

ABSTRACT

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2Kb and H-2Db MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2Kb and H-2Db and the predictive value of these motifs is shortly discussed. The high-affinity H-2Db-binding peptide and putative CTL epitope E7 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E7 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.


Subject(s)
Epitopes/immunology , Papillomaviridae/immunology , Peptide Fragments/immunology , Repressor Proteins , T-Lymphocytes, Cytotoxic/immunology , Tumor Virus Infections/prevention & control , Viral Vaccines/immunology , Amino Acid Sequence , Animals , Cell Line , Cell Line, Transformed , Histocompatibility Antigens Class I/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oncogene Proteins, Viral/immunology , Papillomavirus E7 Proteins , Vaccination
9.
J Virol Methods ; 42(2-3): 265-79, 1993 May.
Article in English | MEDLINE | ID: mdl-8390474

ABSTRACT

Two sets of consensus PCR primers consisting of a common 3' primer CP-I and two 5'-primers, CP-IIG (primer set A) and CP-IIS (primer set B), in the E1 open reading frame of the human papillomavirus (HPV) genome are presented. These two primer sets enabled the detection of a 188 base pair (bp) fragment of HPV 1, 2, 3, 4, 5, 6b, 7, 8, 9, 10a, 11, 12, 14a, 16, 17, 18, 19, 20, 21, 22, 24, 25, 31, 33, 36, 37, 38, 39 and 46. HPV types 15, 23, 49 and 50 were poorly amplified and HPV type 41 was not amplified. The method is suitable for the detection of HPV DNA sequences in clinical samples of both cervical and cutaneous lesions.


Subject(s)
Cervix Uteri/microbiology , DNA, Viral/isolation & purification , Papillomaviridae/isolation & purification , Warts/microbiology , Base Sequence , Consensus Sequence , DNA Probes , DNA, Viral/classification , Female , Humans , Molecular Sequence Data , Papillomaviridae/classification , Papillomaviridae/genetics , Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Homology, Nucleic Acid
10.
J Gen Virol ; 74 ( Pt 1): 121-4, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8380833

ABSTRACT

The human papillomavirus type 16 (HPV-16) enhancer-promoter is virtually inactive in normal human diploid fibroblasts, but active in human fibroblasts with a deletion in the short arm of one chromosome 11 (del-11 cells). Since the HPV-16 enhancer with the simian virus 40 promoter is active in both cell types, the target for chromosome 11-regulated HPV-expression is likely to be located in the HPV-16 early promoter region (nucleotides 57 to 112). We show here that DNA-protein complexes formed with an HPV-16 promoter fragment are quantitatively different in del-11 cell and diploid cell extracts. This quantitative difference detected in band shift experiments disappeared upon mutation of the HPV-16 TATAAAA box to TATTTAT. This mutation also strongly reduced the activity of the HPV-16 enhancer-promoter in del-11 cells. These results indicate that TATA-binding proteins are involved in the chromosome 11-mediated regulation of HPV-16 gene expression.


Subject(s)
Chromosomes, Human, Pair 11/physiology , DNA, Viral/genetics , Gene Expression Regulation, Viral/physiology , Papillomaviridae/genetics , Promoter Regions, Genetic/physiology , Transcription, Genetic/physiology , Base Sequence , DNA, Viral/physiology , Gene Deletion , Gene Expression Regulation, Viral/genetics , Humans , Molecular Sequence Data , Promoter Regions, Genetic/genetics , TATA Box/physiology
11.
EMBO J ; 11(12): 4601-6, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1330540

ABSTRACT

Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.


Subject(s)
Chromosomes, Human, Pair 11 , Gene Deletion , Papillomaviridae/genetics , Phosphoprotein Phosphatases/metabolism , Transcriptional Activation , Amino Acid Sequence , Blotting, Western , Cell Transformation, Viral , Cells, Cultured , Diploidy , Enhancer Elements, Genetic , Ethers, Cyclic/pharmacology , Genes, Viral , Humans , Molecular Sequence Data , Okadaic Acid , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/genetics , Plasmids , Promoter Regions, Genetic , Protein Phosphatase 2 , RNA, Messenger/genetics , Simian virus 40/genetics
12.
J Gen Virol ; 73 ( Pt 12): 3263-8, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1335027

ABSTRACT

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.


Subject(s)
DNA, Viral/analysis , Papillomaviridae/genetics , Tumor Virus Infections/diagnosis , Base Sequence , Cervix Uteri/microbiology , DNA, Viral/genetics , Female , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Papanicolaou Test , Papillomaviridae/classification , Polymerase Chain Reaction , Tumor Virus Infections/genetics , Vaginal Smears
13.
Virology ; 190(1): 40-4, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1326811

ABSTRACT

The human papillomavirus (HPV) type 16 enhancer-promoter has been shown to be active in human fibroblasts with a deletion on the short arm of one chromosome 11 (karyotype 46,del(11)(p11.11p15.1)) but is virtually inactive in diploid human fibroblasts (Smits, Smits, Jebbink, and ter Schegget, 1990b, Virology, 176, 158-165). In diploid human embryonic fibroblasts, activation of the HPV16 enhancer-promoter could be achieved by expression of the SV40 small t. By cotransfecting SV40 small t cDNA together with HPV16 DNA into diploid cells, it was possible to increase the transforming activity of HPV16 by 10- 15-fold. Furthermore, SV40 small t was essential for the SV40 large T-induced morphological transformation of human diploid fibroblasts, whereas SV40 small t was dispensable for transformation of del-11 cells. We propose that, as a result of the deletion of loci on the short arm of chromosome 11 in del-11 cells, functions are expressed that mimic those of SV40 small t in transformation and trans-activation.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Cell Transformation, Viral , Chromosomes, Human, Pair 11 , Papillomaviridae/genetics , Trans-Activators/metabolism , Transcription, Genetic , Cell Line , Chromosome Deletion , Diploidy , Enhancer Elements, Genetic , Humans , Promoter Regions, Genetic
14.
J Gen Virol ; 69 ( Pt 11): 2913-7, 1988 Nov.
Article in English | MEDLINE | ID: mdl-3183632

ABSTRACT

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.


Subject(s)
HIV/genetics , Hot Temperature , Animals , Blotting, Southern , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation , HIV/growth & development , Rats , Repetitive Sequences, Nucleic Acid , Restriction Mapping
15.
J Gen Virol ; 69 ( Pt 6): 1179-93, 1988 Jun.
Article in English | MEDLINE | ID: mdl-2838570

ABSTRACT

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.


Subject(s)
Antigens, Viral/genetics , Arsenites , Cell Cycle , Cytomegalovirus/genetics , Gene Expression Regulation , Immediate-Early Proteins , Sodium Compounds , Viral Proteins/biosynthesis , Acetyltransferases/biosynthesis , Acetyltransferases/genetics , Animals , Antigens, Viral/biosynthesis , Arsenic/pharmacology , Cell Line , Chloramphenicol O-Acetyltransferase , Cycloheximide/pharmacology , Cytomegalovirus/metabolism , DNA, Viral/biosynthesis , DNA, Viral/drug effects , Enhancer Elements, Genetic , Genes, Viral , Hot Temperature , Humans , Nucleic Acid Hybridization , Promoter Regions, Genetic , RNA, Messenger/analysis , RNA, Viral/analysis , Rats , Transcription, Genetic , Transfection
16.
J Gen Virol ; 68 ( Pt 11): 2839-52, 1987 Nov.
Article in English | MEDLINE | ID: mdl-2824664

ABSTRACT

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.


Subject(s)
Cytomegalovirus/genetics , DNA, Viral/genetics , Enhancer Elements, Genetic , Genes, Viral , Methylation , Animals , Antigens, Viral/genetics , Cell Line , Cycloheximide/pharmacology , Cytomegalovirus/growth & development , Gene Expression Regulation/drug effects , Rats , Transcription, Genetic/drug effects , Virus Replication
17.
J Gen Virol ; 68 ( Pt 11): 2925-31, 1987 Nov.
Article in English | MEDLINE | ID: mdl-2824669

ABSTRACT

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.


Subject(s)
Arsenic/pharmacology , Arsenites , Cytomegalovirus/genetics , Gene Expression Regulation , Hot Temperature , Protein Synthesis Inhibitors/pharmacology , Animals , Antigens, Viral/genetics , Cell Line , Cell Nucleus/physiology , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Genes, Viral , In Vitro Techniques , Rats , Transcription, Genetic/drug effects
18.
J Virol ; 58(3): 851-9, 1986 Jun.
Article in English | MEDLINE | ID: mdl-3009892

ABSTRACT

Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.


Subject(s)
Antigens, Viral/biosynthesis , Cytomegalovirus/metabolism , Protein Biosynthesis , Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/genetics , Base Sequence , Cell Line , DNA, Viral/analysis , Fluorescent Antibody Technique , Nucleic Acid Hybridization , Puromycin/pharmacology , Rats , Transcription, Genetic , Transfection , Virus Activation
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