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PubMed1982

Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins.

We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.

Aleutian Mink Disease↗

PubMed1985

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.

Animals↗

PubMed1991

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

Base Sequence↗

PubMed1992

Modification of foot-and-mouth disease virus O1 Caseros after serial passages in the presence of antiviral polyclonal sera.

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability and, like other RNA viruses, presents a high rate of mutation. It has been proposed that selection exerted by antibodies of the host could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant (Nr) populations after serial passages of a cloned FMDV O1 Caseros strain on secondary monolayers of bovine kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages, i.e., 29, under selective pressure, the virus population showed the following characteristics: (i) increased resistance to neutralization by APS (Nr), (ii) altered electrophoretic mobility of its structural viral proteins (VP1), and (iii) alterations at the RNA nucleotide sequence that codes for the major antigenic site of VP1. These acquired characteristics were detected at passage 15 and remained unmodified throughout successive passages. These results document a rapid selection and fixation of specific mutations in response to immunological pressure. In addition, the findings that (i) mutations not related to APS selection were not detected and (ii) after 29 passages at a high multiplicity of infection without immunological pressure, the RNA sequence that codes for VP1 remained unmodified clearly demonstrated that FMDV O1 Caseros presents in vitro a remarkable unexpected genetic stability.

Amino Acid Sequence↗

PubMed1994

Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm.

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.

Amino Acid Sequence↗

PubMed1995

Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B.

Influenza virus infection initiates transcription of a variety of genes for cytokines such as tumor necrosis factor alpha (TNF-alpha), TNF-beta, interleukin 1 alpha, (IL-1 alpha), IL-1 beta, IL-2, IL-4, IL-6, IL-10, granulocyte macrophage colony-stimulating factor, and gamma interferon. However, the mechanism by which virus infection elicits cytokine expression remains unknown. Six influenza virus-induced cytokine genes are targets for the inducible transcription factor NF-kappa B, a central regulator of the human immune response. Here, we show that expression of a single influenza virus protein, the virion surface hemagglutinin, strongly activates NF-kappa B DNA binding and transactivation. Activation is inhibited in the presence of the antioxidant dithiothreitol, suggesting that, similar to the findings for previously described inducers of NF-kappa B, hemagglutinin expression generates radical oxygen intermediates which activate the transcription factor. Hemagglutinin is the first secretory and structural viral protein reported to activate NF-kappa B and thus represents a new class of inducers for this transcription factor. We discuss these results in the context of clinical complications of influenza virus infection.

Antioxidants↗

PubMed1996

Paradoxical interactions between human delta hepatitis agent RNA and the cellular protein kinase PKR.

The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.

Animals↗

PubMed1999

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies.

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.

Animals↗

PubMed2000

Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain.

Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2). A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.

3' Untranslated Regions↗

PubMed2000

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E.

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.

Evolution, Molecular↗

PubMed2002

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes.

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Gamma interferon can block herpes simplex virus type 1 reactivation from latency, even in the presence of late gene expression.

Herpes simplex virus type 1 (HSV-1)-specific CD8+ T cells and the cytokine gamma interferon (IFN-gamma) are persistently present in trigeminal ganglia (TG) harboring latent HSV-1. We define "latency" as the retention of functional viral genomes in sensory neurons without the production of infectious virions and "reactivation" as a multistep process leading from latency to virion assembly. CD8+ T cells can block HSV-1 reactivation in ex vivo mouse TG cultures and appear to be the sole source of IFN-gamma in these cultures. Here we demonstrate that IFN-gamma alone can block HSV-1 reactivation in some latently infected neurons, and we identify points of intervention in the life cycle of the reactivating virus. Cell suspensions of TG that were latently infected with recombinant RE HSV-1 expressing enhanced green fluorescent protein from the promoter for infected cell protein 0 (ICP0) or glycoprotein C (gC) were depleted of endogenous CD8+ or CD45+ cells and cultured in the presence or absence of IFN-gamma. Our results demonstrate that IFN-gamma acts on latently infected neurons to inhibit (i) HSV-1 reactivation, (ii) ICP0 promoter activity, (iii) gC promoter activity, and (iv) reactivation in neurons in which the ICP0 or gC promoter is active. Interestingly, we detected transcripts for ICP0, ICP4, and gH in neurons that expressed the ICP0 promoter but were prevented by IFN-gamma from reactivation and virion formation. Thus, the IFN-gamma blockade of HSV-1 reactivation from latency in neurons is associated with an inhibition of the expression of the ICP0 gene (required for reactivation) and a blockade of a step that occurs after the expression of at least some viral structural genes.

Animals↗

PubMed2005

Deletion of gpUL132, a structural component of human cytomegalovirus, results in impaired virus replication in fibroblasts.

The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratory-adapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.

Base Sequence↗

PubMed2000

Intracellular Virus Trafficking Reveals Physiological Characteristics of the Cytoskeleton.

Virus particles that infect eukaryotic cells can take advantage of the cytoskeleton and associated motors to translocate through the cytoplasm. Depending on the virus, motor proteins are recruited or, alternatively, cytoskeletal elements are induced to polymerize onto viral structures. Here we review recent advances toward understanding the roles of the cytoskeleton in virus trafficking.

Journal Article↗

PubMed2006

Oncolytic capacity of attenuated replicative semliki forest virus in human melanoma xenografts in severe combined immunodeficient mice.

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy. Here, we analyzed the oncolytic potential of the replication-competent vector VA7-EGFP, based on the avirulent Semliki Forest virus (SFV) strain A7(74), to kill cancer cells in culture as well as to target s.c. human melanoma xenografts in severe combined immunodeficient (SCID) mice. VA7-EGFP was able to infect most cancer cell lines studied, leading to complete lysis of the cells within 72 hours after infection. In SCID mice grafted with A2058 human melanoma, marked regression of the xenografts was observed following a single injection of 10(6) plaque-forming units of virus given either i.p., i.v., or intratumorally. Histologic analysis revealed the presence of virus not only in all treated tumors but also in the brains of the treated mice, causing progressing neuropathology beginning at day 16 after infection. Following initial oncolysis, clusters of viable tumor cells were observed embedded in connective tissue, and at later stages, encapsulated tumor nodules had formed. Infection of melanoma cells from explant cultures of these nodules revealed that a portion of the cells were resistant to virus. To be eligible for use in virotherapy, the ability of avirulent SFV to spread within tumor tissue may have to be improved and the biological safety of the virus may have to be addressed thoroughly in higher animals.

Animals↗

PubMedDate not supplied

High doses of methylene blue/light treatment crosslink the A-alpha-subunit of fibrinogen: influence of this photooxidization on fibrinogen binding to platelets.

Irradiation in the presence of 1 microM methylene blue (MB) inactivates enveloped viruses in human plasma. This method is a useful tool to enhance the safety of human fresh-frozen plasma in blood donor banks. Via a photooxidative mechanism, viral structures as well as plasma proteins, especially fibrinogen, can be damaged. We investigated the effect of MB/light-induced photooxidative modification on the structure of fibrinogen and its function in the interaction with platelets in the concentration range of 150 microM MB. Densitometric scanning of reduced SDS-PAGE showed a decrease in the Aalpha-subunit of fibrinogen as well as an increase in high-molecular-weight (HMW) aggregates with increasing MB concentrations. The HMW aggregates may be due to covalent crosslinking of single Aalpha-subunits via photooxidation induced by MB/light treatment. Fibrinogen treated with high doses of MB (50 microM) showed a weaker binding to the fibrinogen receptor (GP IIb/IIIa) on the platelet surface and a decrease in platelet aggregation after stimulation with ADP and photooxidized fibrinogen. These effects were not detected in fibrinogen isolated from virus-inactivated plasma (1 microM MB). This was in agreement with normal binding of fibrinogen to GP IIb/IIIa on platelets.

Adenosine Diphosphate↗

PubMed1980

The pathomorphology of malignant catarrhal fever. I. Generalized lymphoid vasculitis.

A histologic, ultrastructural and immunohistochemical study of the vasculitis in the rete mirabile of 18 calves with experimental and two calves with spontaneous malignant catarrhal fevel showed lesions similar to those reported in graft rejection and some other lymphocyte-associated vasculopathies. The calves were examined at early, mid and late stages of clinical disease. Vascular lesions in the carotid rete were representative of those in other tissues examined. The magnitude of the arterial wall mononuclear cell infiltrate and the degree of arterial wall necrosis and degeneration increased with progression of the clinical disease. The predominant site of mononuclear cell accumulation was the adventitia. There was less medial and intimal involvement. The mononuclear infiltrate was composed of lymphocytes and macrophages with neutrophils and plasma cells rarely present. Endothelial hyperplasia, fibrinoid change and thrombosis were uncommon. Many changes contrasted with those recognized in an active Arthus reaction where adventitial change was minimal and most inflammatory cells were neutrophils or plasma cells. IgG and C3 rarely were detected in vessel walls with fluorescent antibody techniques. Serum immunoglobulins as measured by radial immunodiffusion were not elevated. Viral structures were not seen in tissues examined ultrastructurally.

Animals↗

PubMed1980

The pathomorphology of malignant catarrhal fever. II. Multisystemic epithelial lesions.

Epithelia from 18 Holstein-Fresian calves with experimentally induced malignant catarrhal fever and two calves with naturally occurring disease had severe destructive inflammatory epithelial lesions. Calves were necropsied at early, mid and late clinical stages. Multifocal, degenerative and necrotic epithelial lesions were associated with lymphoid cells. Ultrastructurally, numerous lymphocytes, lymphoblasts and macrophages were accumulated between epithelial cells and in lamina propria, vessels and perivascular tissues. Epithelial cells in such lesions had variably severe degenerative changes or were necrotic but there was no proliferative response. Lesions were widespread in all calves and affected oral, ocular, gastrointestinal, ductal, urinary tract, choroid plexus and other epithelial tissues. Calves killed in late clinical stages had more severe lesions than those killed early. The predominantly lymphocytic, invasive-destructive pattern seen in tissues of cattle with malignant catarrhal fever was similar to that seen in such conditions as contact hypersensitivity and graft versus host disease. Viral structures were not seen.

Animals↗