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At least 433 records · Page 24Linked to original sources

PubMed2003

Binding of adenovirus capsid to dipalmitoyl phosphatidylcholine provides a novel pathway for virus entry.

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Application of crystallography to the design of antiviral agents.

A historical review of x-ray crystallography introduces basic concepts and describes the potential of this science to the rational design of antiviral agents. Following a brief introduction to the study of antiviral compounds that inhibit early stages of rhino- and enteroviral infections, the impact of structural studies on rational drug design is discussed in relation to known viral structures.

Antiviral Agents↗

PubMed2005

Electron microscopy as a reliable tool for rapid and conventional detection of enteric viral agents: a five-year experience report.

BACKGROUND AND AIM OF THE WORK: Since the introduction of the electron microscope and its subsequent development, virology has made a great step forward by the improvement of the basic knowledge on viral structure, as well as by broad application of electron microscopy (EM) to viral diagnosis. In this report, we describe a five-year experience in the use of EM for the diagnosis of enteric viral infections. METHODS: Three thousand four hundred and ninety stool specimens were analyzed at the Virology Unit (Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Italy) during a five-year period, from January 1999 to January 2004. The faecal extracts were subjected to EM after negative staining and were simultaneously cultured to evidence the presence of cytopathogenic agents. RESULTS: EM directly applied to the above specimens allowed the detection of several enteric viral agents, particularly evidencing those normally hard to cultivate (thus easily lost with culture methods). It also enabled diagnosis of dual gut infections, such as those from rotavirus and calicivirus. On the other hand, EM-based identification of viral agents after cell culture and ultracentrifugation of cytopathogenic agent-containing cellular extracts, allowed the identification of cultivable agents, such as picornaviruses, which can escape the direct EM detection if low concentrated. CONCLUSIONS: A rationalized use of EM on selected samples, such as stool, appears suitable in epidemiological or clinical conditions when a very rapid diagnosis is required to save time, including cases of suspected emerging viral infections.

Adenoviruses, Human↗

PubMed1979

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.

Aedes↗

PubMed2000

Augmentation of immune responses to hepatitis E virus ORF2 DNA vaccination by codelivery of cytokine genes.

DNA vaccines encoding a viral structural protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In the present study we show that DNA immunization with a plasmid expressing the hepatitis E virus ORF2 structural protein (pcDNA-ORF2) induced low levels of long-lasting antibody responses in the murine model. The use of plasmids expressing interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating-factor (GM-CSF) in conjunction with pcDNA-ORF2 enhanced the antibody responses generated by pORF-2. We further show that the immune responses generated by plasmid pcDNA-ORF2 can be boosted with virus-like particles composed of the ORF2 protein expressed through a baculovirus expression system.

Animals↗

PubMed1995

IgM anti-hepatitis C virus in patients with chronic non-A, non-B hepatitis and their relationship to viral replication.

Patients with hepatitis C virus (HCV) infection may have different patterns of antibody response to various structural and non-structural viral antigens. We have correlated the serological patterns to the clinical features of chronic infection and to viral replication in 68 HCV-Ab-positive patients with chronic liver disease at different stages (19 with cirrhosis-hepatocellular carcinoma, 38 with chronic active hepatitis and 11 with chronic persistent hepatitis). Serum samples from each patient were assayed for HCV-IgM by enzyme immunoassay and for HCV-RNA by the polymerase chain reaction using primer sets derived from the 5'-non-coding region. The prevalence of HCV-IgM was high (54 patients (79.4%)) and the study showed a good correlation between high values of anti-HCV-IgM and the presence of HCV-RNA in serum, since HCV-RNA was detected in 35 of the 54 IgM-positive patients (64.8%) and notably in 19 of the 20 subjects with high levels of specific IgM. Conversely, all the 35 sera containing HCV-RNA were also reactive for HCV-IgM, while none of the HCV-IgM-negative sera was HCV-RNA reactive. Positivity rates for both HCV-RNA and IgM anti-HCV were higher in the more advanced stages of disease; thus, the clinical pattern of HCV chronic hepatitis seems to be strictly related to the serological pattern and the presence of HCV-RNA.

Journal Article↗

PubMed1979

[Expression of viral antigens on the membrane of normal and leukemic thymocytes of AKR mice].

Expression of antigenous determinants of structural proteins G-MuLV (p10, p12, p30, gp14, gp17) and R-MuLV (gp69/71, p15) on thymocytes of normal and leukemic AKR mice was studied by membrane immunofluorescence. From this sign sharp difference between normal and malignant thymocytes was shown. A possible role in the antitumour immunity of antigens to structural viral proteins MuLV expressed on the leukemic cell membrane is discussed.

Animals↗

PubMed1993

Cytomegalovirus IgM antibody detection: comparison of five assays.

Human Cytomegalovirus (CMV) infection can result in a dramatic disease in immunosuppressed patients. For effective treatment sensitive and specific procedures are required for prompt and early diagnosis of active CMV infection. In the present study five different serological assays were used for detection of CMV-specific immunoglobulin M antibodies (IgM) in 78 sera, obtained from patients with a clinical suspicion of CMV infection. In addition, sequential sera, obtained from renal transplant recipients who experienced a primary CMV infection, were analysed by the five IgM detection assays. The assays used were two enzyme-linked immunosorbent assays (ELISA): the Vironostika CMV ELISA from Organon Teknika (ELISA-1) and the Monosan CMV ELISA from Sanbio (ELISA-2), the CMV microparticle enzyme immunoassay (MEIA) from Abbott and two Western-(immuno)blotting techniques using purified CMV structural viral proteins of the AD169 strain (IB-AD169) and the major non structural DNA binding protein of 52 kiloDalton (IB-Rp52) as antigenic material. The results of the assays were compared to each other with respect to sensitivity, specificity and overall agreement, as well as incidence of false positive and false negative results. Although the MEIA gave the highest sensitivity, the ELISA-1 combined a high sensitivity with a high specificity and therefore appears the most suitable method for determination of active CMV infection.

Adolescent↗

PubMed2005

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains.

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into non-immunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.

Animals↗

PubMed1993

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Amino Acid Sequence↗

PubMed1983

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line.

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.

Cell Line↗

PubMed1999

Identification of three major phosphorylation sites within HIV-1 capsid. Role of phosphorylation during the early steps of infection.

We previously reported the presence of two cellular serine/threonine protein kinases incorporated in human immunodeficiency virus type 1 (HIV-1) particles. One protein kinase is MAPK ERK2 (mitogen-activated protein kinase), whereas the other one, a 53-kDa protein, still needs to be identified. Furthermore, we demonstrated that the capsid protein CAp24 is phosphorylated by one of those two virion-associated protein kinases (Cartier, C., Deckert, M., Grangeasse, C., Trauger, R., Jensen, F., Bernard, A., Cozzone, A., Desgranges, C., and Boyer, V. (1997) J. Virol. 71, 4832-4837). In this study, we showed that CAp24 is not a direct substrate of MAPK ERK2. Moreover, using site-directed mutagenesis of each of the 9 serine residues of CAp24, we demonstrated the phosphorylation of 3 serine residues (Ser-109, Ser-149, and Ser-178) in the CAp24. Substitution of each serine residue did not affect viral budding, nor viral structure. By contrast, substitution of Ser-109, Ser-149, or Ser-178 affects viral infectivity by preventing the reverse transcription process to be completely achieved. Our results suggest that CAp24 serine phosphorylation is essential for viral uncoating process.

Amino Acid Substitution↗

PubMed2002

HIV-1-associated dementia: a metabolic encephalopathy perpetrated by virus-infected and immune-competent mononuclear phagocytes.

Infection of the nervous system by HIV-1 commonly causes a broad range of cognitive, behavioral, and motor abnormalities called, in its most severe form, HIV-1-associated dementia (HAD). HAD is a metabolic encephalopathy caused by productive viral infection of brain mononuclear phagocytes (MPs) (perivascular and parenchymal brain macrophages and microglia) and sustained by paracrine-amplified, inflammatory, neurotoxic responses. MP neurotoxins are, in large measure, homeostatic secretory products that can have a negative effect on neuronal cell function when produced in abundance. Proinflammatory cytokines, chemokines, platelet-activating factor, arachidonic acid and its metabolites, nitric oxide, quinolinic acid, progeny virions, and viral structural and regulatory proteins are all included as part of these cellular and viral toxic elements. In addition, neuronal damage can occur directly by engaging specific receptors or through inducing widespread inflammatory activities in brain tissue that ultimately induce neuronal demise. The mechanisms for immune-and viral-mediated neural injury in HAD are made more striking by the effects of abused drugs on cognitive function. Ultimately, linkages between neuronal function and disordered MP immunity will provide insights into how HIV-1 infection of the brain leads to compromised mental function as well as providing clues into the pathogenesis of other neurodegenerative disorders.

AIDS Dementia Complex↗

PubMed2005

The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells.

An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid-November 2002. The aetiological agent of this disease was found to be a previously unknown coronavirus, SARS-associated coronavirus (SARS-CoV). The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein, which is predicted to be a transmembrane protein. In this study, it was shown that the 3a protein was expressed in the lungs and intestinal tissues of SARS patients and that the protein localized to the endoplasmic reticulum in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggested that the 3a protein may trigger apoptosis. These data showed that overexpression of a single SARS-CoV protein can induce apoptosis in vitro.

Animals↗

PubMed1996

The rev-responsive element negatively regulates human immunodeficiency virus type 1 env mRNA expression in primate cells.

The human immunodeficiency virus type 1 (HIV-1) Rev protein mediates the accumulation of unspliced and singly spliced viral transcripts within the cytoplasm of infected cells, late in the infection cycle, leading to the expression of the viral structural proteins, Gag, Pol, and Env. Rev binds to a complex RNA structure, the Rev-responsive element (RRE), present in all Rev-responsive viral transcripts, relieving their nuclear sequestration. The precise mechanism by which RRE-containing transcripts are retained within the nucleus in the absence of Rev protein is not well understood. We previously demonstrated that the RRE alone plays a crucial role in the nuclear retention of RRE-containing env transcripts in stably transfected Drosophila cells. Here we extend our previous observations and demonstrate that the RRE is a principal determinant of nuclear retention for envelope transcripts in primate cells and, in particular, human CD4+ T cells.

Animals↗

PubMed1983

Disproportionate synthesis of Semiliki Forest virus 42S and 26S RNAs in polyamine-depleted baby hamster kidney cells.

The role of polyamines in viral RNA synthesis has been studied using Semliki Forest virus-infected, polyamine-depleted baby hamster kidney cells as a model system. The synthesis of viral 42S RNA, which corresponds to the viral genome, was markedly inhibited, while the synthesis of viral 26S RNA, which acts as a messenger for viral structural proteins, was reduced much less or not at all. The decreased total viral RNA synthesis and the ratio of 42S to 26S RNA were rapidly returned to normal by adding spermidine to the culture medium. From these results it can be hypothesized that polyamines have a special role in the synthesis of viral RNA, possibly affecting the conformation of the RNA template.

Animals↗

PubMed1997

A dynamic in vivo view of the HIV-I Rev-RRE interaction.

The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

Base Sequence↗

PubMed2006

Phosphatidylserine is involved in gene expression from Sindbis virus subgenomic promoter.

Sindbis virus replication is mediated by an RNA replicase translated from viral RNA genome. The replicase synthesizes progeny genomic RNA and shorter RNA (subgenomic RNA) carrying viral structural genes in association with cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine (PS), in Sindbis virus gene expression using Chinese hamster ovary cell mutants. When the mutant cells were transfected with viral replicon RNA, in which the structural genes downstream of the subgenomic promoter were replaced by a reporter gene, reporter expression was inhibited under PS-deficient conditions. In contrast, reporter expression from an SV40 promoter-driven construct was normal under similar conditions. Furthermore, expression of a viral replicase protein from the genomic RNA and accumulation of the subgenomic RNA were not inhibited by PS deficiency. These findings indicate that reduced cellular PS level impairs a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression.

Animals↗