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PubMed1989

Replication of measles virus in cultured human thymic epithelial cells.

Measles virus can replicate in cultures of both infantile and fetal human thymic epithelial cells. Virus-induced cytopathology including syncytium formation was first evident around 24 hr after viral inoculation of these cultures. At the same time, the cultures began to lose their characteristic thymus-like organizational structure. Viral antigens were detected in infected cells by indirect immunofluorescence, and the presence of progeny virions was demonstrated in culture fluids.

Animals↗

PubMed1982

A deficiency in the repair of UV and gamma-ray damaged DNA in fibroblasts from Cockayne's syndrome.

The host-cell reactivation of V antigen production for irradiated adenovirus was examined in fibroblasts from 5 unrelated patients with Cockayne's syndrome (CS) and 2 CS heterozygotes. The fibroblast cultures were infected with either irradiated or non-irradiated adenovirus and subsequently examined for the presence of viral structural antigens using immunofluorescent staining. All CS-homozygous strains showed a reduced host-cell reactivation (HCR) of this viral function for both UV-and gamma-irradiated virus. For UV-irradiation of the virus, D37 values expressed as a percentage of that obtained on normal strains, ranged from 14 to 35%. For gamma-irradiation of the virus these values ranged from 61 to 80%. These results indicate some defect in the repair of both UV- and gamma-ray-induced DNA damage for CS. 1 CS-heterozygote strain tested also showed a reduced HCR for UV-irradiated adenovirus intermediate between that of the patient strain and normal, whereas another CS-heterozygote strain showed apparently normal HCR level.

Adenoviruses, Human↗

PubMed1984

Characterization of the effect of rimantadine on intracellular distribution and transport into the nuclei of infected cells of the parent influenza virus genome.

Rimantadine in the concentration of 50 micrograms/ml did not affect the distribution in subcellular fractions of MDCK cells of the parent influenza virus A/Khabarovsk/74/77(H1N1) labelled with 14C-amino acids, 3H-nucleosides or 32P-orthophosphoric acid. In the presence of rimantadine viral structures of heterogenous composition were detected in the nuclei of infected cells. Their buoyant densities of 1.34-1.36, 1.41-1.42 and 1.28-1.32 g/cm3 in the CsCl gradient were characteristic for RNP and "nucleoids" (complex of RNP and M protein), respectively. Similar structures were also detected in the nuclei of cells infected in the absence of rimantadine. No primary transcription of the viral genome was registered in the presence of rimantadine, although the genetic material in the nuclei was present in a form suitable for transcription.

Adamantane↗

PubMed1995

Human retroviruses and demyelinating diseases.

The consequences of human retroviral infections have had an unprecedented impact on the medical, scientific, and social institutions of the last two decades of this century. The nervous system as an end target organ figures prominently in the constellation of diseases associated with HTLV and HIV infection and numerous syndrome complexes have been recognized that reflect dysfunction of the brain, spinal cord, nerve roots, peripheral nerves, or muscle. HAM/TSP, associated with HTLV-I and rarely with HTLV-II infections, and encephalomyelitis, associated with HIV infection, may present with clinical, laboratory, neuroelectrophysiologic, and neuroimaging features closely resembling MS. A careful systematic search for associated disease processes and review of the medical history, however should raise the suspicion of possible retroviral infection. In the appropriate setting, because of the pleiotropy in disease expression and the high prevalence of retroviral infection in many areas of the United States, clinicians should have a low threshold for ordering diagnostic testing for HIV and HTLV when considering a retroviral cause for a neurologic disorder. The retroviruses are pervasive throughout the vertebrate subphylum and share common elements within their genome that encode for promoters and structural proteins and are distinguished from each other by sets of transactivating and regulating genes. The latter group of genes serve to regulate viral replication by the induction and post-transcriptional and post-translational modification of retrovirally encoded gene products. In addition, the transactivating gene products can activate and upregulate the expression of a variety of host cellular genes, many of which possess immune-related functions. The viral particle or specific components of certain viral structural proteins may be directly toxic to neural tissues. Also, during the replicative phase, retroviruses are recognized by host defense mechanisms, which mount considerable cellular and humoral immune responses. The spectrum of retroviral-associated neurologic diseases therefore may represent a complex interaction among viral antigen-induced immunity, the production of neurotoxic viral peptides, and the abnormal induction and expression of cellular genes with potent bioactivity.

HIV Infections↗

PubMed1975

Replication of eastern equine encephalitis viruses (New Jersey and Louisiana strains and the Ets-4 mutant) in rabbit kidney cells.

Virus yield, viral RNA synthesis, viral protein synthesis, cytopathology, and virus-induced shutoff of host protein synthesis were examined in rabbit kidney (RK) cells infected with eastern equine encephalitis (EEE) viruses. The New Jersey (NJ) strain replicated most rapidly, and exhibited a distinct patter in the synthesis of structural and non-structural viral proteins. The Louisiana (LA) strain and the Ets-4 mutant appeared to be similar in RK celles and chick embryo fibroblasts. Although the viral peptides comprising the viral RNA polymerase could not be conclusively identified, two non-structural proteins of molecular weights 105,000 and 85,000 were temporally associated with increasing rates of polymerase activity in NJ-infected RK cells and were found in increased amounts in Ets-4-infected cells compared to La-infected cells.

Animals↗

PubMed1994

Adenovirus inhibition of cell translation facilitates release of virus particles and enhances degradation of the cytokeratin network.

Infection of animal cells by a number of viruses generally results in an array of metabolic defects, including inhibition of host DNA, RNA, and protein synthesis, and morphological alterations known as cytopathic effects. For adenovirus infection there is a profound loss of cell structural integrity and a marked inhibition of host protein synthesis, the latter generally assumed necessary to enhance virus production. We examined the purpose of viral inhibition of cell translation and found that it was related in part to cytopathic wasting of infected cells. We show that viral shutoff of host translation promotes destruction of the intermediate filament network, particularly cytokeratins which are proteolysed at keratins K7 and K18 by the adenovirus late-acting L3 23-kDa proteinase. We found that if adenovirus is prevented from inhibiting cell translation, the intermediate filament network remains relatively intact, keratin proteins are still synthesized, and cells possess an almost normal morphological appearance and lyse poorly, reducing the release of nascent virus particles by several hundredfold. Remarkably, in tissue culture cells the accumulation of late viral structural proteins is only marginally reduced if host translation shutoff does not occur. Thus, a surprising major function for adenovirus inhibition of cellular protein synthesis is to enhance impairment of cellular structural integrity, facilitating cell lysis and release of progeny adenovirus particles.

2-Aminopurine↗

PubMed2005

Stoichiometric limitations in assembly of active recombinant retrovirus.

Although recombinant retroviruses are widely used in gene therapy and as gene transfer vehicles for basic biological studies, their titers are very low as compared to other recombinant viral systems, e.g., adenovirus. We investigated the rate-limiting steps in production of LacZ-encoding ecotropic (CRE BAG 2) and amphotropic (Psi-CRIP) retrovirus. We found that ecotropic retrovirus producer cells produced a large number of inactive viral particles because they were severely limited by the amount of mRNA that was packaged into viral capsids. Introduction of the gene for green fluorescence protein (GFP) increased retroviral titers 40-fold, without affecting the viral matrix protein, p30, or the activity of reverse transcriptase. Surprisingly, while transfer of GFP gene increased retrovirus production, beta-gal activity and X-gal titer decreased significantly. Quantitative real-time polymerase chain reaction (PCR) showed that although producer cells synthesized similar amounts of both mRNAs, retroviral supernatants contained significantly lower amount of LacZ mRNA, possibly due to competition between LacZ and GFP mRNAs for encapsidation into virions. In contrast to ecotropic producers, introduction of GFP gene copies into amphotropic producers resulted in a moderate twofold increase in retrovirus production. However, delivery of genes encoding for the viral proteins gp70 and p30 increased virus production by fivefold, suggesting that amphotropic producers may also be limited by synthesis of structural viral proteins. Our data show that in addition to the amount of viral genome or proteins, assembly of viral components into active viral particles may limit production of high titer retroviral preparations.

Capsid↗

PubMed1994

Detection of bovine viral diarrhoea virus p80 protein in subpopulations of bovine leukocytes.

Flow cytometry and two-colour immunofluorescence were used to detect cytoplasmic bovine viral diarrhoea virus (BVDV) antigen in leukocytes from viraemic cattle. Monoclonal antibody to the p80 protein of BVDV, a non-structural viral antigen, was used to identify the subpopulations of leukocytes in which viral protein synthesis had occurred. Viral antigen was detected in 23% of peripheral blood mononuclear cells. Monocytes were found to have the highest frequency of infection (35%). A higher proportion of CD2+ T cells (23%) were infected, compared with B cells (11%) or WC1+ gamma delta T cells (11%). No significant differences in percentages of different leukocyte subpopulations in blood were detected in persistently viraemic animals compared with controls.

Animals↗

PubMed1978

Intracellular synthesis of measles virus-specified polypeptides.

The intracellular synthesis of measles-specified polypeptides was examined by means of polyacrylamide gel electrophoresis of cell extracts. Since measles virus does not efficiently shut off host-cell protein synthesis, high multiplicities of infection were used to enable viral polypeptides to be detected against the high background of cellular protein synthesis. The cytoplasm of infected cells contained viral structural polypeptides with estimated molecular weights of 200,000, 80,000, 70,000, 60,000, 41,000, and 37,000. All of these structural polypeptides, with the exception of P1, the only virion glycoprotein (molecular weight congruent to 80,000), were also found in the nuclei. In addition, two nonstructural polypeptides with estimated molecular weights of 74,000 and 72,000 were also present in the cytoplasm of infected cells. The initial synthesis of the smaller, nonstructural polypeptide began later in infection than the structural polypeptides. Pulse-chase experiments failed to detect any precursor-product relationships. The intracellular glycosylation and phosphorylation of the viral polypeptides were found to be similar to those found in purified virions.

Cell Line↗

PubMed1998

Identification and functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter.

The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3' end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902-3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.

Base Sequence↗

PubMed1990

Virology of the human immunodeficiency virus type 1.

Understanding the mode of replication of the human immunodeficiency virus (HIV) is necessary for explaining the natural history of HIV infection and for the development of therapeutic and prophylactic agents. The life cycle of HIV involves infective particles, the attachment of the virus to specific cell receptors, reverse transcription of the viral RNA into DNA by using viral encoded enzymes, integration of the viral DNA into the host genome, use of the host machinery to transcribe and translate viral genes, and the formation of budding particles. Viral encoded genes, regulated by a complex interaction of viral and host proteins with viral sequences, ultimately encode for the viral structural proteins, regulatory proteins, and enzymes necessary for the formation of mature infectious particles. Blocking one or more steps in the viral life cycle and preventing enhancement of virus production are important approaches in the control of HIV.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis.

Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of virions.

Base Sequence↗

PubMed1984

Relation of HVJ (Sendai virus) production to cell growth phase in persistently infected mouse 3T3 cells.

Using 3T3 mouse fibroblasts persistently infected with temperature-sensitive HVJ (3T3-HVJ pi), the relationship between production of HVJ and cell growth was investigated. In these cells the highest virus release into the culture media occurred from 24 to 48 hours after seeding. The release gradually decreased as the cultures approached confluency, at which time it was reduced below 1-2 per cent of that on day 2 in parallel with the decrease of both cellular DNA and RNA synthesis rates. Comparative examination in growing and resting HVJ carrier 3T3 cells showed that amounts of nucleocapsids and rate of viral structural protein synthesis in the latter phase was reduced to 4-5 per cent of those in the former phase. In addition, viral replication and transcription rates in the resting phase were found to be suppressed to 8-9 per cent of levels detected in the growing phase. These results suggest that the reduced virus production in the resting HVJ carrier cells may be mainly due to the suppression of viral RNA synthesis.

Animals↗

PubMed2004

Antiviral activity of lactoferrin towards naked viruses.

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

Adenoviridae↗

PubMed1997

A protoplast system for studying tomato spotted wilt virus infection.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

Animals↗

PubMed1988

Use of Ar+ plasma etching to localize structural proteins in viruses: studies with adenovirus 2.

The experiments described here were undertaken to test the idea that low energy Ar+ plasma etching could be employed as the basis of a method to order viral structural polypeptides according to their physical proximity to the virus surface. Since low energy (500 eV) Ar+ ions do not penetrate deeply into virus surfaces, one expects that the outermost proteins will be damaged before internal ones when intact virions are irradiated. To test this expectation, we exposed adenovirus 2 to a 500-eV Ar+ plasma and then employed sodium dodecyl sulfate-polyacryl-amide gel electrophoresis to assess the extent of damage to the major structural polypeptides. Gel analyses showed that the proteins exposed on the virus surface (proteins II, III, and IV) were degraded rapidly during the first 10 s of irradiation while protein VII, the major core polypeptide, was almost completely protected. Proteins located between the capsid and the core, such as proteins IIIa and VI, were degraded at intermediate rates. Quantitative measurements demonstrated that the observed decay rate differences were not due simply to differences in protein target size; distance to the virion surface made an important contribution. The plasma etching technique, therefore, appears to have considerable potential for the structural analysis of viruses and other macromolecular assemblies where the proximity of individual proteins to the particle surface is unknown.

Adenoviridae↗

PubMed2005

Sheep retrovirus structural protein induces lung tumours.

Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.

Animals↗

PubMed1975

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

Cells, Cultured↗