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Molecular basis of sickle cell-endothelial cell interactions.

Adherence of sickle erythrocytes to microvascular endothelium is posited to initiate or contribute to sickle cell vaso-occlusive pain episodes. Adherence and occlusion in vivo may depend on hemodynamics interacting with plasma, erythrocyte, and endothelial cell factors. Four receptor-mediated adherence pathways have been described to date: adherence mediated by high molecular weight von Willebrand factor multimers bridging glycoprotein lb-like and integrin receptors on sickle cells and similar receptors on endothelial cells; thrombospondin bridging CD36 on sickle reticulocytes and the alpha v beta 3 integrin on large-vessel endothelial cells or alpha v beta 3 and CD36 on microvascular endothelium; binding of sickle reticulocyte alpha 4 beta 1 receptors to vascular cell adhesion molecule 1 expressed on endothelial cells stimulated by cytokine or double-stranded RNA viruses; and binding of sickle cells to endothelial cell-associated fibronectin via sickle reticulocyte alpha 4 beta 1 activated by phorbol ester or interleukin-8. The significance of these adherence pathways in sickle cell vaso-occlusion is discussed.

Endothelium, Vascular↗

Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography.

Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 A (1.5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.

Birnaviridae↗

Micromonas pusilla reovirus: a new member of the family Reoviridae assigned to a novel proposed genus (Mimoreovirus).

Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90-95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV.

Amino Acid Sequence↗

Protection of agammaglobulinemic piglets from porcine rotavirus infection by antibody against simian rotavirus SA-11.

Rotavirus, a double-stranded RNA virus, has been implicated as a diarrhea-provoking agent in a variety of animal species. Several previous reports have shown that immunization with a single serotype may result in increased in vitro neutralization titers against serotypes not represented in the immunogen. This study was undertaken to determine whether antibody from cows immunized against simian rotavirus strain SA-11 (which is alien to pigs) could protect neonatal piglets from infection with a North Carolina isolate of porcine rotavirus. Accordingly, cows were immunized with SA-11 and an immunoglobulin G (IgG)-rich fraction was isolated from their colostrum. An IgG-rich fraction was similarly isolated from colostrum of nonimmunized cows. At equal concentrations, IgG from SA-11-immunized cows had two- to fourfold higher neutralization titers to seven of eight test strains of rotavirus, including SA-11 (serotype 3); human rotavirus serotypes 1, 3, and 4; North Carolina porcine rotavirus (serotype undetermined); Ohio State porcine rotavirus (serotype 5); and bovine rotavirus (serotype 6). The IgG-rich fractions were fed as dietary supplements to agammaglobulinemic piglets infected with the North Carolina porcine rotavirus. IgG from the SA-11-immunized cows was about eightfold more effective in protecting piglets than was IgG from nonimmunized cows.

Agammaglobulinemia↗

Circulating immunoglobulin G can play a critical role in clearance of intestinal reovirus infection.

Reoviruses are encapsidated double-stranded RNA viruses that cause systemic disease in mice after peroral (p.o.) inoculation and primary replication in the intestine. In this study, we define components of the immune system involved in the clearing of reovirus from the proximal small intestine. The intestines of immunocompetent adult CB17, 129, and C57BL/6 mice were cleared of reovirus serotype 3 clone 9 (T3C9) within 7 days of p.o. inoculation. Antigen-specific lymphocytes were important for the clearance of intestinal infection, since severe combined immunodeficient (SCID) mice failed to clear T3C9 infection. To define specific immune components required for intestinal clearance, reovirus infection of mice with null mutations in the immunoglobulin M (IgM) transmembrane exon (MuMT; B cell and antibody deficient) or beta 2 microglobulin gene (beta 2-/-; CD8 deficient) was evaluated. beta 2-/- mice cleared reovirus infection with normal kinetics, while MuMT mice showed delayed clearance of T3C9 7 to 11 days after p.o. inoculation. Adoptive transfer of splenic lymphocytes from reovirus-immune CB17 mice inhibited growth of T3C9 in CB17 SCID mouse intestine 11 days after p.o. inoculation. The efficiency of viral clearance by adoptively transferred cells was significantly diminished by depletion of B cells prior to adoptive transfer. Results in SCID and MuMT mice demonstrate an important role for B cells or IgG in clearance of reovirus from the intestines. Polyclonal reovirus-immune rabbit serum, protein A-purified immune IgG, and murine monoclonal IgG2a antibody specific for reovirus outer capsid protein sigma 3 administered intraperitoneally all normalized clearance of reovirus from intestinal tissue in MuMT mice. This result demonstrates an IgA-independent role for IgG in the clearance of intestinal virus infection. Polyclonal reovirus-immune serum also significantly decreased reovirus titers in the intestines of SCID mice, demonstrating a T-cell-independent role for antibody in the clearance of intestinal reovirus infection. B cells and circulating IgG play an important role in the clearance of reovirus from intestines, suggesting that IgG may play a more prominent functional role at mucosal sites of primary viral replication than was previously supposed.

Animals↗

Mutational analysis of the role of nucleoside triphosphatase P4 in the assembly of the RNA polymerase complex of bacteriophage phi6.

Bacteriophage phi6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.

Bacteriophage phi 6↗

The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae.

We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth.

Adaptor Proteins, Signal Transducing↗

Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein.

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

Baculoviridae↗

Ski6p is a homolog of RNA-processing enzymes that affects translation of non-poly(A) mRNAs and 60S ribosomal subunit biogenesis.

We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5'-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)- viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.

Amino Acid Sequence↗

Kinetics of ribosomal pausing during programmed -1 translational frameshifting.

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.

Base Sequence↗

Molecular diversity of partial-length genomic segment 2 of human picobirnavirus.

OBJECTIVES: This study was carried out in order to evaluate the efficacy of the recently developed picobirnavirus (PBV) sets of primers and to establish the phylogenetic relationships of Argentine strains with PBV strains isolated in China and the USA. METHODS: Thirteen fecal specimens tested as positive for PBV by polyacrylamide gel electrophoresis were analyzed by reverse transcription-polymerase chain reaction assays using primers target to the genomic segments 2 of PBV strains isolated in China and the USA. The amplicons were sequenced and analyzed. RESULTS: Primers derived from the China strain produced amplicons in only 4 of the 13 specimens (30.76%). No sample was revealed as positive with the primers derived from the US strain. DNA sequencing of polymerase chain reaction products differed in nucleic acid and amino acid sequences by 13.9-42.28% and 18.1-51.1%, respectively. Despite this strain diversity, three domains of conserved nucleotide sequences as well as the amino acid motif D-S-D typical of RNA-dependent RNA polymerase gene of double-strand RNA viruses were identified. Comparatively, these conserved regions were also identified in homologous PBV strains from the USA and China. Phylogenetic analysis showed no time or geographic clustering. CONCLUSIONS: These findings provide evidence that PBV may represent an emerging heterogeneous group of viruses.

Adolescent↗

The role of interferon-alpha in a successful murine tumor therapy.

Combination therapy using reovirus type 3 and the chemo-therapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is sufficient to cure approximately 80% of EL-4 lymphoma tumor-bearing BD2F1 male mice. Cured animals can be challenged with the EL-4 tumor, in the absence of the therapy, to yield 100% survival, whereas those challenged with heterologous tumor produce 0% survival. These results strongly suggest that a host-immune response is responsible for the observed therapeutic effect. Reovirus, a double-stranded RNA virus, is an efficient inducer of type I interferon. In an effort to determine the role of virus in this therapy, we substituted interferon-alpha (IFN-alpha) for reovirus in the therapy. Doses of IFN-alpha from 1000-10,000 U were capable of replacing reovirus to produce cure rates similar to reovirus. Spleen cells isolated from therapy-treated animals demonstrated high levels of cytotoxicity against the natural killer cell-sensitive cell line YAC-1, but not against EL-4 tumor. In vitro stimulation of isolated spleen cells by IFN-alpha resulted in a high level of natural killer cell activity, but no cytotoxicity against the EL-4 tumor. A significant antiproliferative effect against the EL-4 tumor in cell culture was demonstrated by IFN-alpha. Finally, therapy-treated, tumor-bearing mice that were injected with anti-IFN-alpha + -beta antibodies had similar survival levels as control mice, indicating that other cytokines might also play a role in promoting tumor killing. These investigations suggest that IFN-alpha may be a mediator of antitumor activity in the reovirus therapy system.

Animals↗

Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

Animals↗

Short report: detection of Leishmaniavirus in human biopsy samples of leishmaniasis from Peru.

Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.

Animals↗

Short report: quantification of leishmaniavirus RNA in clinical samples and its possible role in pathogenesis.

Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.

Adolescent↗

Control and eradication of African horse sickness with vaccine.

African horse sickness (AHS) is an infectious but no-contagious viral disease of equidae with high mortality in horses. The disease is caused by an arthropod-borne double-stranded RNA virus within the genus Orbivirus of the family Reoviridae transmitted by at least two species of Culicoides. Nine different serotypes have been described. The nine serotypes of AHS have been described in eastern and southern Africa. Only AHS serotypes 9 and 4 have been found in West Africa from where they occasionally spread into countries surrounding the Mediterranean. Examples of outbreaks that have occurred outside Africa are: in the Middle East (1959-1963), in Spain (serotype 9, 1966, serotype 4, 1987-1990), and in Portugal (serotype 4, 1989) and Morocco (serotype 4, 1989-1991). Laboratory diagnosis of AHS is essential. Although the clinical signs and lesions are characteristic, they can be confused with those of other diseases. Several techniques have been adapted for the detection of RNA segments, antibodies and antigen. Two types of vaccines have been described for AHS virus. Attenuated live vaccines (monovalent and polyvalent) for use in horses, mules and donkeys, are currently available, as well as a monovalent, serotype 4, inactivated vaccine, produced commercially but no longer available. New vaccines, including a subunit vaccine, have been evaluated experimentally. In this paper a review of the last AHS outbreaks in Spain, occurring during 1987-1990, and affecting the central and south part of the country, is presented. The role that vaccination played for the control and eradication of the disease, as well as other aspects such as climatological conditions, number of vectors and horse management, are also presented and evaluated.

African Horse Sickness↗

Association of yeast viral 23 S RNA with its putative RNA-dependent RNA polymerase.

T double-stranded RNA (dsRNA) and its single-stranded RNA (ssRNA) counterpart, 23 S RNA, are present in some strains of the yeast Saccharomyces cerevisiae. 23 S RNA is identical with the T (+)-strand, and both are cytoplasmically inherited. The T (+)-strand encodes a protein of 104 kilodaltons (kDa) (p104) that has sequences diagnostic of RNA-dependent RNA polymerases from (+)-strand and double-stranded RNA viruses. Here we show that p104 is expressed only in yeast strains carrying T dsRNA (and 23 S RNA) and that the ability to produce p104 is co-transmitted from one strain to another by cytoplasmic mixing (cytoduction) along with T dsRNA. Neither T nor 23 S RNA is encapsidated into viral particles; however, the latter was found to be non-covalently associated with p104. This finding was based on the evidence that: (i) both 23 S RNA and p104 cosedimented in sucrose gradients and that (ii) 23 S RNA was immunoprecipitated by a p104-specific polyclonal antiserum. Consistent with this association, p104 shows single-stranded RNA-binding activity in Northwestern blots. We have localized the RNA-binding domain of p104 within its C terminus 234 amino acids.

Fungal Proteins↗

Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia.

Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.

Animals↗