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PubMed2006

Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans.

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.

Animals↗
PubMed1985

Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication.

Animals↗
PubMed1991

Infectious entry pathway of adenovirus type 2.

Internalization of the infectious fraction of human adenovirus type 2 into HeLa cells was followed by a quantitative internalization assay. Treatments known to selectively block receptor-mediated endocytosis reduced the internalization of infectious virus to an extent close to the reduction of endocytosis of transferrin. This suggests that one of the first steps in the infectious cycle of adenovirus type 2 is internalization by the coated-pit and -vesicle pathway.

Adenoviruses, Human↗
PubMed2001

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Bacteriophage PRD1↗
PubMed2002

Differential transmission of human immunodeficiency virus type 1 by distinct subsets of effector dendritic cells.

Dendritic cells (DC) support human immunodeficiency virus type 1 (HIV-1) transmission by capture of the virus particle in the mucosa and subsequent transport to the draining lymph node, where HIV-1 is presented to CD4(+) Th cells. Virus transmission involves a high-affinity interaction between the DC-specific surface molecule DC-SIGN and the viral envelope glycoprotein gp120 and subsequent internalization of the virus, which remains infectious. The mechanism of viral transmission from DC to T cells is currently unknown. Sentinel immature DC (iDC) develop into Th1-promoting effector DC1 or Th2-promoting DC2, depending on the activation signals. We studied the ability of these effector DC subsets to support HIV-1 transmission in vitro. Compared with iDC, virus transmission is greatly upregulated for the DC1 subset, whereas DC2 cells are inactive. Increased transmission by DC1 correlates with increased expression of ICAM-1, and blocking studies confirm that ICAM-1 expression on DC is important for HIV transmission. The ICAM-1-LFA-1 interaction is known to be important for immunological cross talk between DC and T cells, and our results indicate that this cell-cell contact is exploited by HIV-1 for efficient transmission.

Cell Communication↗
PubMed1981

[Morphology of coronaviruses-- electron microscopic demonstration by negative contrast technic of the transmissible gastroenteritis virus of swine].

This contribution to the morphology of corona virus is based on the use of electron microscopy in a negative contrast investigation of the virus of transmissible gastroenteritis of swine. Viruses of transmissible gastro-enteritis are spherical or, in rare cases, pleomorphous particles of typical corona virus structure. The viruses investigated by the authors were 132 nm in diameter. The surface projections, consisting of a flattened knob and conical shaft, are 18 nm in length. Internal bodies, 50 +/- 2 nm in diameter, occur as "filled" or "empty" particles. The thickness of all internal bodies and virus membranes is 8 +/- 1 nm. Differences were found to exist between virus of transmissible gastro-enteritis and particles similar to corona virus and isolated from faecal samples. A first comparison had been made for that purpose. Those differences were relating to shape and size of individual particles and to morphological setup of surface projections.

Animals↗
PubMed2006

Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

Cytomegalovirus↗
PubMed2003

Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter.

Based on the observation that an internally located 3' promoter sequence can be functional (R. Flick and G. Hobom, Virology, 1999, 262(1), 93-103), we generated transfectant influenza A viruses harboring a dicistronic segment containing the CAT gene (660 nt) or a fragment of the Mengo virus VP0 capsid gene (306 nt) under the control of a duplicated 3' promoter sequence. Despite slightly reduced NA expression, the transfectant viruses replicated efficiently and proved to be stable upon both serial passage in vitro in MDCK cells and in vivo replication in the pulmonary tissue of infected mice. Internal initiation of replication and transcription from the second, internal, 3' promoter directed the synthesis of subgenomic vRNA and mRNA and therefore permitted expression of the foreign gene product, e.g., the CAT enzyme. The design of this vector may prove particularly appropriate for the utilization of influenza virus for the expression of heterologous proteins in their native form.

3' Untranslated Regions↗
PubMed1988

Humoral and cytotoxic T cell immune responses to internal and external structural proteins of simian virus 5 induced by immunization with solid matrix-antibody-antigen complexes.

Monoclonal antibodies (MAbs) were complexed to solid matrices and used to purify virus proteins from simian virus 5 (SV5)-infected tissue culture cells, ideally in such a way as to bind equimolar amounts of antigen to antibody. The resulting solid matrix-antibody-antigen (SMAA) complexes were then used as immunogens and successfully induced specific humoral and cytotoxic T cell responses. By attaching more than one MAb to the solid matrix and using such complexes to purify the respective proteins multivalent immunization was achieved. Analysis of the cytotoxic T cell response of immunized animals indicated that both surface and internal SV5 structural proteins can act as target antigens. Immunization with SMAA complexes, in the absence of adjuvant, induced higher levels of antibody than the antigen alone precipitated on alum. However, immunization with SMAA complexes resulted in relatively less antibody being produced to the antigenic determinant through which the protein is coupled, via antibody, to the SMAA complex compared with the amount of antibody produced against other antigenic determinants on that protein. The particulate solid matrix used to form the SMAA complexes in most of the experiments was a 'fixed' and killed suspension of the Cowan A strain of Staphylococcus aureus, although preliminary results indicated that Protein A-Sepharose could also be used as a solid matrix. Prior immunization with S. aureus alone did not reduce the level of the immune response to the appropriate antigen on subsequent immunization with S. aureus-antibody-antigen complexes. In fact on immunization of mice with these complexes the level of antibody produced to the S. aureus matrix itself was less when S. aureus-antibody-antigen complexes were used as immunogens than when S. aureus or S. aureus-antibody complexes were used. Furthermore, rabbits immunized with S. aureus-mouse MAb-antigen complexes showed a vigorous immune response to the antigen.

Animals↗
PubMed1984

Genome structure and expression of a defective interfering mutant of the killer virus of yeast.

A large internal deletion in M1 double-stranded (ds) RNA from the killer virus of Saccharomyces cerevisiae generates a suppressive (S3) dsRNA molecule. Strains which harbor S3 dsRNA are defective in toxin production and immunity to the toxin. The biochemical defect in expression has been investigated and is apparently due to truncation of the protoxin polypeptide translation reading frame on S3 dsRNA. Transcription in vivo, and in isolated virions in vitro, results in the synthesis of a full-length positive polarity messenger RNA, denoted s. The s transcript contains no long poly(A) tracts as determined by its lack of affinity for oligo(dT)-cellulose, and as inferred by sequence analysis of approximately 87% of the S3 dsRNA genome. These data support a model for template coding of polyadenylate in transcripts derived from the wild-type M1 dsRNA. The orientation of the sequences conserved on S3 dsRNA with respect to M1 dsRNA has been determined. Some of the conserved sequences are likely to be required for the maintenance and replication of these viral dsRNA genomes in S. cerevisiae.

Amino Acid Sequence↗
PubMed1991

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses.

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.

Animals↗
PubMed2005

Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp.

The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

Animals↗
PubMed1989

Modulation of lentivirus replication by antibodies. Non-neutralizing antibodies to caprine arthritis-encephalitis virus enhance early stages of infection in macrophages, but do not cause increased production of virions.

Non-neutralizing antibodies to caprine arthritis-encephalitis virus (CAEV) enhance the early stages of the virus life cycle but do not potentiate enhanced production of virus particles by macrophages. In primary macrophages used for these studies, there was enhancement in binding, internalization and uncoating of virus pretreated with non-neutralizing sera in comparison to virus pretreated with a non-immune serum. However, this did not lead to enhanced production of virus particles. Failure of non-neutralizing sera to inactivate CAEV may be due in part to low avidity of the antibodies for the virus particles which contain sialic acids on their envelopes, because desialylation of the particles made them neutralizable. The non-neutralizing antibodies probably bound to most of the native virus particles which were then internalized via Fc receptor-mediated endocytosis and degraded. Sialylated particles that failed to bind antibodies probably caused the infection. Thus there was no true enhancement of infection. The previously reported increase in severity of lesions in animals immunized with inactivated CAEV particles prior to challenge with live virus suggested enhancement of infection but in the light of our finding this may have been caused by factors other than an increase in production in the number of infectious virus particles.

Animals↗
PubMed1977

Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus.

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of l-[(35)S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (gag) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with d-[(3)H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the gag proteins and that several of these polyproteins are glycosylated. A comparison of gag precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.

Cell Cycle↗
PubMed2000

Translation initiation of a bicistronic mRNA of Borna disease virus: a 16-kDa phosphoprotein is initiated at an internal start codon.

We examined translational initiation of a bicistronic 0.8-kb mRNA of Borna disease virus (BDV) using a cDNA clone of the mRNA. Upon transfection with the clone, COS-7 cells produced a 16-kDa protein (P'), in addition to the previously identified products of BDV, 24- (P) and 14.5-kDa proteins. The 16-kDa product was detected by anti-P monoclonal antibody and was shown to exist in BDV-infected cell lines as well as in infected animal brain cells. Transient expression analysis of mutated cDNA clones encoding the BDV 0.8-kb mRNA revealed that the 16-kDa protein was initiated at the second AUG codon on the same open reading frame of the P protein. The mutational analysis also demonstrated that the first AUG within the 0.8-kb mRNA is not optimal, although the signal contains a better Kozak's motif. These results demonstrated the presence of three functional AUG codons in the smallest mRNA of BDV and also suggested that a leaky scanning mechanism is involved in translational initiation at AUG codons downstream of the bicistronic mRNA of BDV. Furthermore, the 16-kDa protein was located in the BDV-specific nuclear foci and was found to associate with the other viral proteins in BDV-infected cells, demonstrating an important role of the novel identified BDV protein in viral replication.

Amino Acid Sequence↗
PubMed1993

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.

Animals↗