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At least 559 records · Page 31Linked to original sources
PubMed1997

Infection of blood and bone marrow cells with the human cytomegalovirus in vivo.

The human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. Both, primary infection and reactivation of latent virus can cause disease. Peripheral blood leukocytes (PBL) most likely play an important role in viral persistence and dissemination of infection. However, an open question has been whether HCMV actively replicates in PBL in vivo and whether the progenitor cells in the bone marrow are also infected. Previous studies on this issue are controversial. Here we summarize data on the tropism of HCMV for mature leukocyte populations as well as bone marrow progenitor cells during HCMV viremia. All cell populations were highly purified by a fluorescence activated cell sorter (FACS) and analyzed by PCR for the presence of viral genomic DNA. Moreover, mature leukocyte populations were investigated for mRNA expression of regulatory and viral structural proteins. We could show, that HCMV DNA was detected most frequently in granulocytes and monocytes as well as in CD34+ progenitor cells of immunosuppressed patients. Viral mRNA expression was found in granulocytes, monocytes, and lymphocyte fractions. In contrast, no HCMV DNA was found in healthy, seropositive individuals.

Bone Marrow↗
PubMed1992

Structural and functional analysis of the visna virus Rev-response element.

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.

Base Sequence↗
PubMed1983

Characterization of Gypsy Moth (Lymantria dispar) Nuclear Polyhedrosis Virus.

Characterization of the proteins and nucleic acid of the gypsy moth nuclear polyhedrosis virus isolated in Ithaca, N.Y. (LdNPV-IT) is presented. A total of 29 viral structural proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the virus was isolated in the absence of alkaline protease activity. Fourteen surface envelope viral proteins were identified by lactoperoxidase iodination. Eleven proteins were associated with nucleocapsids prepared by Nonidet P-40 detergent treatment. Distinct alterations of viral proteins were documented when virions were purified in the presence of occlusion body-associated alkaline protease(s). Restriction enzyme digests of viral DNA indicated that this isolate was composed of a large number of genetic variants. On the basis of the major molar fragments resulting from EcoRI, BamHI, BglII, and HindIII digests, the molecular weight of the LdNPV genome was approximately 88 x 10.

Journal Article↗
PubMed1981

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.

Adenoviridae↗
PubMed1978

The retrovirus particles in human myeloma cells RPMI8226: morphological, biochemical, immunological and infective transmission studies.

The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as its propagation in various animal and human cells. The myeloma cells RPMI8226 produce intracytoplasmatic A-type particles and extracellular particles. The extracellular particles have been classified as immature particles with translucent core center, typical mammalian C-type virus particles and C-type particles with intermediate membrane. However, the budded particles in secondarily infected human neoplastic cells contained complete doughnut-shaped nucleoids. This type of budding is rather characteristic for B-type particles. The 3H-uridine labeled RPMI8226 viral particles have a buoyant density 1.17 g/ml in sucrose gradient containing high molecular weight RNA and the distribution of viral structural proteins in SDS-PAGE is characteristic for oncornaviruses. The internal structural proteins according to MW are ranged from 13 000 to 30 000 daltons. The virus contains a magnesium-dependent reverse transcriptase. The cellular homogenate and viral concentrate from RPMI8226 cultures do not react with antibodies against ALSV, MuLV, FeLV, RD114, MP-MV and SiSLV. The only reaction was scored with anti BLV antibodies. However, anti BLV serum inhibiting the reverse transcriptase activity of BLV to 60% does not cross-react with the reverse transcriptase of RPMI8226V. In contrast to BLV concentrates, neither XC nor KC cells show syncytia formation by RPMI8226V. The RPMI8226V replication is restricted to human tumor and normal human glia-like cells. The possible origin of the virus is discussed.

Antigens, Viral↗
PubMed1994

A single stem-loop structure within the HTLV-1 Rex response element is sufficient to mediate Rex activity in vivo.

The human T-cell leukemia virus (HTLV-1) Rex protein is required for the cytoplasmic expression of the incompletely spliced transcripts that encode the viral structural proteins. This effect is mediated by a highly structured cis-acting RNA element of 254 nucleotides termed the Rex response element, or RexRE. Here we demonstrate that one of the four known RexRE stem-loop structures as well as a 43-nt segment derived from this element is sufficient to mediate Rex function in vivo. Upon duplication, this stem-loop is shown to function as efficiently as the full-length RexRE. In vitro RNA binding analyses with wildtype and mutagenized RNA show that this stem-loop contains a high affinity binding site for Rex that coincides with a predicted bulge structure in the central part of this stem-loop. These results indicate that a small region of the RexRE containing a high affinity binding site is sufficient to mediate Rex function and suggest that sequences outside of this binding site have no unique role in mediating Rex regulation.

Base Sequence↗
PubMed1977

Use of a radioimmune assay in detection of measles antibodies in cerebrospinal fluid and serum.

Evidence that different structural components of the measles virus may act as antigens has been demonstrated by the serologic methods of hemagglutination inhibition, hemolysin inhibition and nucleocapsid complement fixation. Using radioiodinated measles viral antigens, an immune precipitation assay has been designed which is capable of discriminating varying reactivities to measles viral structural components in serum or CSF and of differentiating whether IgG and IgM antibody is involved. This technic has been applied to the study of measles antibodies in CSF and sera of patients with multiple sclerosis (MS) and other neurologic diseases. From data presented here, it is found that both groups of patients have individual reactivity to measles proteins, present in CSF and serum, while three normal CSF sample were not found to have such antibodies.

Antibodies, Viral↗
PubMed1987

Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus.

Recombinant DNA techniques were used to delete regions of a cDNA clone of the phosphoprotein NS gene of vesicular stomatitis virus. The complete NS gene and four mutant genes containing internal or terminal deletions were inserted into a modified pGem4 vector under the transcriptional control of the phage T7 promoter. Run-off transcripts were synthesized and translated in vitro to provide [35S]methionine-labeled complete NS or deletion mutant NS proteins. Immune coprecipitation assays involving these proteins were developed to map the regions of the NS protein responsible for binding to the structural viral nucleocapsid protein N and the catalytic RNA polymerase protein L. The data indicate the NS protein is a bivalent protein consisting of two discrete functional domains. Contrary to previous suggestions, the negatively charged amino-terminal half of NS protein binds to L protein, while the carboxyl-terminal half of NS protein binds to both soluble recombinant nucleocapsid protein N and viral ribonucleocapsid template.

Amino Acid Sequence↗
PubMed2004

Evidence for a polytopic form of the E1 envelope glycoprotein of Hepatitis C virus.

The polyprotein precursor of the Hepatitis C virus (HCV) contains multiple membrane-spanning domains that define the membrane topology and subsequent maturation of the viral structural proteins. In order to examine the biogenesis of the E1-E2 heterodimeric complex, we inserted an affinity tag (S-peptide) at specific locations within the envelope glycoproteins. In particular, and based on the prediction that the E1 glycoprotein may be able to assume a polytopic topology containing two membrane-spanning domains, we inserted the affinity tag within a putative cytoplasmic loop of the E1 glycoprotein. The HCV structural polyprotein containing this tag (at amino acids 295/296) was highly expressed and able to form a properly processed and noncovalently associated E1-E2 complex. This complex was bound by murine and conformation-dependent human monoclonal antibodies (MAbs) comparably to the native untagged complex. In addition, MAb recognition was retained upon reconstituting the tagged E1-E2 complex in lipid membrane as topologically constrained proteoliposomes. Our findings are consistent with the model of a topologically flexible E1 glycoprotein that is able to adopt a polytopic form. This form of the E1-E2 complex may be important in the HCV life cycle and in pathogenesis.

Amino Acid Sequence↗
PubMed1975

Detection of measles antibodies in cerebrospinal fluid and serum by a radioimmunoassay.

Evidence that different structural components of the measles virus may act as antigens has been provided by the serologic methods of hemagglutination inhibition hemolysin inhibition, and nucleocapsid complement fixation. Using radioiodinated measles viral antigens, and immune precipitation assay has been designed that is capable of discriminating among various reactivities to measles viral structural components in serum or cerebrospinal fluid (CSF) and of distinguishing whether IgG and IgM antibody is involved. This technique has been applied to the study of measles antibodies in CSF and sera of patients with multiple sclerosis (MS) and other neurologic diseases. From data presented here, it was found that both groups of patients have individual reactivity to measles proteins, present in CSF and serum, whereas three normal CSF samples were found not to have such antibodies. It appears that oligoclonal immunoglobulins in CSF of MS patients may be detected by this method, and one patient with MS was found to have CSF IgM anti-measles antibodies.

Antibodies, Viral↗
PubMed1999

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Amino Acid Sequence↗
PubMed2006

Superiority of infectivity-based over particle-based methods for quantitation of drug resistant HIV-1 as inocula for cell cultures.

Performance of phenotypic assays and replication capacity assays require normalization of virus input. Therefore, quantitation of HIV-1 in supernatants to inoculate cell cultures is an important step. Since the gold standard for the determination of infectivity, the tissue culture infectious dose 50% (TCID50) is time-consuming, several other methods are in use. This study evaluated methods for the quantitation of drug resistant viruses in cell culture supernatants. The compared methods were based on the detection of viral structural components like genomic RNA or p24 antigen (CA-p24) (particle-based), the determination of reverse transcriptase (RT) activity, and methods based on the detection of viral infectivity like LTR-induced beta-galactosidase (beta-gal) activity and the TCID50 (infectivity-based). Significant correlations were observed between beta-gal activity and TCID50, and between CA-p24 and viral RNA. RT activity did not correlate with any other method. However, RT activity correlated significantly with infectivity when non-resistant subtype-B isolates were analyzed. In contrast to viral infectivity, CA-p24 exhibited a long half life and accumulated in cell culture, resulting in decreasing ratios of infectious virions to CA-p24 over time. As a consequence, relative replication capacities of drug resistant viruses were only determined reliably if the input virus was normalized according to infectivity. In conclusion, RT activity seems to be feasible for non-resistant subtype-B viruses but may be of limited use for non-B subtypes and for drug resistant viruses. Methods determining infectivity are most suitable for quantitation of cell culture inocula, whereas particle-based assays are more appropriate for quantitation of virus production during an experiment.

Anti-HIV Agents↗
PubMed2004

Hepatitis C virus core protein regulates p300/CBP co-activation function. Possible role in the regulation of NF-AT1 transcriptional activity.

Hepatitis C virus (HCV) core is a viral structural protein; it also participates in some cellular processes, including transcriptional regulation. However, the mechanisms of core-mediated transcriptional regulation remain poorly understood. Oncogenic virus proteins often target p300/CBP, a known co-activator of a wide variety of transcription factors, to regulate the expression of cellular and viral genes. Here we demonstrate, for the first time, that HCV core protein interacts with p300/CBP and enhances both its acetyl-transferase and transcriptional activities. In addition, we demonstrate that nuclear core protein activates the NH2-terminal transcription activation domain (TAD) of NF-AT1 in a p300/CBP-dependent manner. We propose a model in which core protein regulates the co-activation function of p300/CBP and activates NF-AT1, and probably other p300/CBP-regulated transcription factors, by a novel mechanism involving the regulation of the acetylation state of histones and/or components of the transcriptional machinery.

Acetylation↗
PubMed1983

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.

Base Sequence↗
PubMed1997

Inhibitory effects of chronic ethanol consumption on cellular immune responses to hepatitis C virus core protein are reversed by genetic immunizations augmented with cytokine-expressing plasmids.

Chronic hepatitis C viral (HCV) infection is a major clinical problem in alcoholics with liver disease and may result from ethanol effects on the host immune response. To experimentally assess such effects, the DNA-based immunization approach was used to produce humoral and cellular immune responses against the HCV core protein in mice. Mice were fed an ethanol or isocaloric pair-fed control liquid diet followed by immunizations with HCV core DNA-expressing constructs. Chronic ethanol feeding was found to inhibit Th cell and CTL activities and substantially reduced cytokine secretion as well. In addition, a switch from Th1 to Th0 subtype was observed in proliferating CD4+ T cells derived from chronic ethanol-fed mice. These immunosuppressive effects were directly due to ethanol since crossover experiments to an isocaloric control diet restored the defects in cellular immunity. Furthermore, we determined if coadministration of an IL-2 or GM-CSF DNA expression plasmid with a plasmid expressing the HCV core protein (pHCV2-2) would reverse the inhibitory effects of chronic ethanol feeding on cellular immune responses. Coimmunization of chronic ethanol-fed mice with either IL-2 or GM-CSF expression plasmids restored cellular immunity and induced CD4+ inflammatory T cell and CD8+ CTL responses comparable with control mice immunized with pHCV2-2 alone. These studies provide evidence of how chronic ethanol feeding may effect cellular immune responses to a viral structural protein in the context of genetic immunization.

Adjuvants, Immunologic↗
PubMed2000

Influenza virus matrix protein is the major driving force in virus budding.

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.

Animals↗
PubMed1984

In vitro translation of infectious flacherie virus RNA in a wheat germ and a rabbit reticulocyte system.

Infectious flacherie virus is an insect picornavirus isolated from the silkworm, Bombyx mori. Its RNA was found to act as an efficient mRNA in a wheat germ extract and a rabbit reticulocyte lysate translation system. In either system the sum of molecular weights of translation products far exceeded the coding capacity of the virus genome, which suggests the occurrence of proteolytic cleavage of large primary products to smaller polypeptides as reported for other picornaviruses and/or premature termination of translation. The highest molecular weight product of 200 000 (polyprotein-like product) could be translated in both systems. One of the antigenic products common to both systems had a molecular weight of 130 000, which corresponds to the sum of molecular weights of the four major viral proteins. Another product, which comigrated with viral protein 0, the largest viral structural protein in SDS-polyacrylamide gel electrophoresis, also showed antigenicity. Peptide mapping of these polypeptides showed that the two in vitro systems translated the same cistron in the viral RNA and that the smaller polypeptide was a part of the 130 000 Da product.

Animals↗