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PubMed1989

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells.

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus.

Adenoviruses, Human↗

PubMed1996

[Assembly of JC virus pseudovirus particles by recombinant DNA in COS7 cells].

JS virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system would play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SR alpha 296 were used to express JC viral structural genes. VP231-SR alpha, which encodes VP2/VP3 and VP1, but lacks 137bp of the 5'-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SR alpha transfected cells. In this study, evidence for JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis and serological diagnosis, are discussed.

Cell Line, Transformed↗

PubMed1990

Characterization of immunostimulating complexes (ISCOMS) of HIV-1.

HIV-1, strain HTLV-III, propagated in H9 cells and purified by sucrose gradient centrifugation, was used as native antigen source for the preparation of immunostimulating complexes, HIV-iscoms. The major antigen detected in the iscom was the cell-derived HLA-DR, which readily could be removed from the virus lysate by immunosorbent. In the iscoms the HIV structural proteins MA p17, p55 and TM gp41 were identified; SU gp120 was present in only minute amounts in the virus lysate. The iscom particles appeared well preserved after freeze drying with a round shape, approximately 35 nm in diameter, comprising morphological subunits, assembled with icosahedral symmetry. Immunization experiments in mice reflected the antigen content of the iscoms. High antibody response was induced to HLA-DR in non-depleted iscoms. Major humoral responses were observed to the viral structural proteins MA p17, CA p24, p55, and also to TM gp41. A low or negligible antibody response to SU gp120 was induced by the HIV-iscoms. The negligible response was, however, overcome by the addition of recombinant gp160 to the virus lysate prior to formation of iscoms, resulting in a preparation evoking a clear serum antibody to gp160.

Animals↗

PubMed1981

Comparative analysis of RNA genomes of mouse hepatitis viruses.

The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.

Animals↗

PubMed1985

Clonal selection of T lymphocytes infected by cell-free human T-cell leukemia/lymphoma virus type I: parameters of virus integration and expression.

We have successfully transmitted cell-free HTLV-I to normal cord blood and peripheral blood lymphocytes and have exploited this system to study the kinetics of infection and transformation of these cells. Transmission was successful in 4 out of 23 attempts. In all 4 cases, the infected cells progressed from an initial stage of polyclonality to predominantly monoclonal cells within 4-6 weeks. Both complete and defective proviruses were transmitted to the recipient cells initially, but cells with a complete provirus were preferentially maintained. The monoclonally infected cells have persisted in culture for more than 6 months and may be considered immortalized. Expression of core antigens as detected by immunoflourescence and the reverse transcriptase activity in the medium at least in one case was not observed until weeks after the cells had become monoclonal, suggesting that expression of virus or viral structural proteins is not necessary for selected growth of the infected cells in vitro.

Bone Marrow Cells↗

PubMed2006

Development of human monoclonal antibodies against diseases caused by emerging and biodefense-related viruses.

Polyclonal antibodies have a century-old history of being effective against some viruses; recently, monoclonal antibodies (mAbs) have also shown success. The humanized mAb Synagis (palivizumab), which is still the only mAb against a viral disease approved by the US FDA, has been widely used as a prophylactic measure against respiratory syncytial virus infections in neonates and immunocompromised individuals. The first fully human mAbs against two other paramyxoviruses, Hendra and Nipah virus, which can cause high (up to 75%) mortality, were recently developed; one of them, m101, showed exceptional potency against infectious virus. In an amazing pace of research, several potent human mAbs targeting the severe acute respiratory syndrome coronavirus S glycoprotein that can affect infections in animal models have been developed months after the virus was identified in 2003. A potent humanized mAb with therapeutic potential was recently developed against the West Nile virus. The progress in developing neutralizing human mAbs against Ebola, Crimean-Congo hemorrhagic fever, vaccinia and other emerging and biodefense-related viruses is slow. A major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies, is the viruses' heterogeneity and mutability. A related problem is the low binding affinity of crossreactive antibodies able to neutralize a variety of primary isolates. Combinations of mAbs or mAbs with other drugs, and/or the identification of potent new mAbs and their derivatives that target highly conserved viral structures, which are critical for virus entry into cells, are some of the possible solutions to these problems, and will continue to be a major focus of antiviral research.

Antibodies, Monoclonal↗

PubMed1992

Host cell reactivation of gamma-irradiated adenovirus 5 in human cell lines of varying radiosensitivity.

DNA repair processes play an important role in the determination of radiation response in both normal and tumour cells. We have investigated one aspect of DNA repair in a number of human cell lines of varying radiosensitivity using the adenovirus 5 host cell reactivation assay (HCR). In this technique, gamma-irradiated virions are used to infect cells and the ability of the cellular repair systems to process this damage is assayed by a convenient immunoperoxidase method recognising viral structural antigen expression on the cell membrane 48 h after infection. Reduced HCR was exhibited by radioresistant HeLa cells and by a radiosensitive neuroblastoma cell line, HX142. In contrast, an ataxia telangiectasia cell line, AT5 BIVA, did not show reduced HCR. On the basis of these results we can make no general conclusions about the relevance of HCR to cellular radiosensitivity. We have extended these studies to determine whether our cell lines exhibited enhanced viral reactivation (ER) following a small priming dose of gamma-radiation given to the cells before viral infection. No evidence for this phenomenon was found either in normal or tumour cell lines.

Adenoviruses, Human↗

PubMed1979

Decreased repair of gamma-irradiated adenovirus in Xeroderma pigmentosum fibroblasts.

The ability of gamma-irradiated adenovirus to produce viral structural antigens (Vag) was examined in several normal and Xeroderma pigmentosum (XP) fibroblast strains. The fibroblast cultures were infected with either irradiated or nonirradiated adenovirus and at 48 hours after infection, cells were examined for the presence of Vag using immunofluorescent staining. Survival of Vag synthesis for gamma-irradiated adenovirus had a D37 value of 47 +/- 4 x 10(4) rad following the infection of seven normal fibroblast strains. The survival of this viral function was found to be significantly less following infection of the XP strains. D37 values for Vag synthesis expressed as a percentage of that obtained on normal strains were obtained for a representative strain from each of the XP complementation groups: group A, 57 per cent; group B, 61 per cent; group C, 61 per cent, group D, 59 per cent; group E, 73 per cent; and variant, 75 per cent. These results indicate that XP cells have a reduced repair capacity for some type of gamma-ray-induced DNA damage.

Adenoviridae↗

PubMed2005

Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.

Animals↗

PubMed1991

Transient suppression of equine immune responses by equine infectious anemia virus (EIAV).

Suppression of the immune system is a common aspect of the disease pathogenesis associated with retroviral infections in both man and animals. We have measured transient suppression of the equine immune system as a loss or decrease in antigen-specific and polyclonal lymphocyte proliferation following experimental infection of ponies with three variants of equine infectious anemia virus (EIAV) with difference virulence characteristics. The transient suppression of proliferative responses was temporally associated with recurrent febrile episodes, which are the hallmark symptom of EIAV-induced disease. Decreased proliferative responses occurred at all times when EIAV viremia was identified, based on the detection of an infectious virus in plasma or viral proteins on peripheral blood mononuclear cells. The immunosuppression was observed most frequently in ponies infected with virulent variants of EIAV which suggested that this effect may contribute to disease pathogenesis. Suppression of polyclonal proliferative responses was induced in vitro by the addition of either infectious or heat-inactivated EIAV to cultures, demonstrating that the viral structural proteins were immunosuppressive in the absence of infection. These studies indicated that EIAV is similar to other retroviruses in that it has the ability to suppress the immune system.

Animals↗

PubMed1983

Some immunochemical aspects of HD-brain extracts.

Cortical gray matter extracts of patients with Huntington's disease (HD) were investigated for their antigenic properties with serum from both HD patients and normal controls. Both patient and control sera appeared to precipitate two closely migrating proteins of MW 14.4K and 15K, which could be found in normal brain preparations only at much lower concentrations, as precipitation of these proteins could not effectively be inhibited by a concentrated "cold" normal brain extract. It is shown that these molecules are hemoglobin and lysozyme, which to a certain extent bind specifically to the immunoprecipitates. The increase in hemoglobin level, which amounted to about 20% of the total protein concentration, might be explained either as an epiphenomenon to the disease process, in which degeneration of the HD brain leads to a relative increase of the arterial and capillary blood system, or as an artifact due to the preparation of the brain extracts. The lysozomal increase is a feature shared by many degenerative neurological diseases. No measurable humoral reactivity could be demonstrated in patient sera against a presumed mutant HD-specific protein. In addition, using antisera to the viral structural protein p30, no evidence could be found for the involvement of a retrovirus in HD.

Adult↗

PubMed2006

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines.

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.

Calcium↗

PubMed1999

Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins.

It has previously been demonstrated in this laboratory that an influenza virus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins from recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliver an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be altered drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression of NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 and NS1 proteins dramatically decreased transmission of the CAT RNA to the MDCK cultures. These results are discussed with reference to the roles of these proteins during virus replication. From these experiments, a ratio of transfected plasmids was found that increased the efficiency of the previously described system by 50-100-fold. Under these optimized conditions, it was demonstrated that VLPs can be formed in the absence of neuraminidase expression and that these VLPs remained aggregated to each other and to cell membranes. Moreover, it is shown that CAT RNA transmission was dependent on specific interactions of the ribonucleoprotein complex with other viral structural polypeptides. These data demonstrate the usefulness of this encapsidation-packaging system for the study of different aspects of the influenza virus life-cycle.

Animals↗

PubMed1975

LCM virus infection of cells in vitro.

Most mammalian cells cultivated in vitro can be infected with lymphocytic choriomeningitis (LCM) virus. In addition to infectious virus, the cells produce antigenic material that fixes complement in the presence of antibody and is precipitated by antiserum. Intracellular antigen can also be demonstrated by the immunofluorescence procedure. When infected cells are viewed with the electron microscope, viral structures are seen either budding from or in association with the cell membranes. Immunoelectron microscopy, immunofluorescence, and cytotoxicity tests reveal virus-specific antigens on the surface of intact cells. Virus multiplication may be succeeded by cytolysis. Two LCM virus-specific antigens (or antigenic groups) can at present be distinguished. One corresponds to the infectious virus; the other is the complement-fixing "soluble" antigen. This extractable complement-fixing activity is produced by infected cells and is also a structural component of the infectious virus. It is not represented on the surface of either the virion or the infected cell. The cytolytic potential of LCM virus varies and is dependent on its previous passage history. Cytolytic and "attenuated" variants are able to initiate persistent infection of Mus musculus.Together with infectious virus, particles are produced that temporarily protect cells against standard virus. They appear to be by-products of virus multiplication, not in the sense of deletion mutants but of virus structures insufficiently equipped for their own active or passive replication, though capable of interfering with infectious virus. No evidence has been found for the generation of "defective interfering" particles, though their presence has not yet been excluded.

Animals↗

PubMed1990

Studies on vaccination against papillomaviruses: a comparison of purified virus, tumour extract and transformed cells in prophylactic vaccination.

Calves were vaccinated with two preparations made from one cutaneous fibropapilloma induced by bovine papillomavirus type 2 (BPV-2). One vaccine consisted of homogenised tumour; the other contained purified virus only. Both produced resistance to a heavy challenge infection of BPV-2. One calf in the vaccinated group developed a small tumour and rejected it earlier than the control calves. It would appear likely that the prophylactic immune response was induced by viral structural proteins only and that tumour-specific antigens are unnecessary. Bovine fibroblasts were transformed in vitro by BPV-2 and administered as a vaccine; immunity was not induced.

Animals↗

PubMed1988

Physicochemical properties of Marburg virus: evidence for three distinct virus strains and their relationship to Ebola virus.

The physicochemical and antigenic properties of three groups of Marburg (MBG) virus isolates, separated temporally and geographically, were compared to each other and to another member of the same family, Ebola (EBO) virus. Each MBG isolate contained seven virion proteins, one of which was a glycosylated surface protein. Peptide mapping of glycoproteins, nucleoproteins (NP) and viral structural protein (VP40) demonstrated extensive sequence conservation in the proteins of viruses isolated over a 13-year period, but homology was not evident in VP24. Some homology between the NPs of MBG and EBO was observed. A close antigenic relationship between MBG strains was found by radioimmunoassay but no evidence was found of antigenic cross-reactivity with EBO viruses. MBG virion proteins are produced from virus-specific monocistronic mRNA species. Five of the seven viral proteins were produced by in vitro translation of these RNAs. MBG virions contained one RNA species with an Mr of 4.2 x 10(6) and virions had a density of 1.14 g/ml in potassium tartrate. Virus isolates from different outbreaks had distinct T1 oligonucleotide maps, but had approximately 95% homology in base sequence. No two geographically distinct virus pairs were more closely related to each other than to a third virus isolate. MBG viruses are thus similar to EBO viruses in morphology and other physicochemical properties and are very similar to each other in RNA and protein composition. Each of the three geographically and temporally distinct MBG virus outbreaks appears to have been due to a genetically distinguishable, but antigenically closely related virus strain. In addition, these studies confirm the belief that MBG and EBO viruses are members of the new virus family, the Filoviridae.

Animals↗

PubMed2006

Two modes of herpesvirus trafficking in neurons: membrane acquisition directs motion.

Alphaherpesvirus infection of the mammalian nervous system is dependent upon the long-distance intracellular transport of viral particles in axons. How viral particles are effectively trafficked in axons to either sensory ganglia following initial infection or back out to peripheral sites of innervation following reactivation remains unknown. The mechanism of axonal transport has, in part, been obscured by contradictory findings regarding whether capsids are transported in axons in the absence of membrane components or as enveloped virions. By imaging actively translocated viral structural components in living peripheral neurons, we demonstrate that herpesviruses use two distinct pathways to move in axons. Following entry into cells, exposure of the capsid to the cytosol resulted in efficient retrograde transport to the neuronal cell body. In contrast, progeny virus particles moved in the anterograde direction following acquisition of virion envelope proteins and membrane lipids. Retrograde transport was effectively shut down in this membrane-bound state, allowing for efficient delivery of progeny viral particles to the distal axon. Notably, progeny viral particles that lacked a membrane were misdirected back to the cell body. These findings show that cytosolic capsids are trafficked to the neuronal cell body and that viral egress in axons occurs after capsids are enshrouded in a membrane envelope.

Animals↗

PubMed1989

Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.

Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp gag-pol sequence with ACTGCCC repeat, a 113-bp mid-pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse species showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus, all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

Animals↗