pinecone/core-2020-05-10-deduplication huggingface dataset:
Comparative Analysis of the Ability of the Polymerase Complexes of Influenza Viruses Type A, B and C to Assemble into Functional RNPs that Allow Expression and Replication of Heterotypic Model RNA Templates In Vivo

AbstractInfluenza viruses type A, B, and C are human pathogens that share common structural and functional features, yet they do not form natural reassortants. To determine to what extent type-specific interactions of the polymerase complex with template RNA contribute to this lack of genotypic mixing, we investigated whether homotypic or heterotypic polymerase complexes support the expression and replication of model type A, B, or C RNA templates in vivo. A plasmid-based expression system, as initially described by Pleschka et al. [(1996) J. Virol. 70, 4188–4192] for influenza A virus, was developed for influenza viruses B/Harbin/7/94 and C/Johannesburg/1/66. The type A core proteins expressed heterotypic model RNAs with similar efficiencies as the homotypic RNA. The influenza B virus model RNA was efficiently expressed by all three types of polymerase complexes. Although no functional polymerase complex could be reconstituted with heterotypic P protein subunits, when the influenza A virus P proteins were expressed together with heterotypic nucleoproteins, significant, albeit limited, expression of RNA templates of all influenza virus types was detected. Taken together, our results suggest that less strict type-specific interactions are involved for the polymerase complex of influenza A compared with influenza B or C viruses
Naturally Occurring, Nonregressing Canine Oral Papillomavirus Infection: Host Immunity, Virus Characterization, and Experimental Infection

AbstractPapillomaviruses occasionally cause severe, nonregressing or recurrent infections in their human and animal hosts. The mechanisms underlying these atypical infections are not known. Canine oral papillomavirus (COPV) typically regresses spontaneously and is an important model of mucosal human papillomavirus infections. A severe, naturally occurring, nonregressing COPV infection provided an opportunity to investigate some aspects of viral pathogenicity and host immunity. In this case, the papillomas proved refractory to surgical and medical treatments, including autogenous vaccination and vaccination with capsid (L1) virus-like particles. High levels of induced anti-L1 antibodies appeared to have no effect on the infection. The papillomas spread to oesophageal mucosa, perioral haired skin, and remote cutaneous sites. Isolation of COPV from the animal and sequencing of several regions of the viral genome showed no differences to the COPV prototype. Experimental infection of beagle dogs with this viral isolate resulted in the uncomplicated development and regression of oral warts within the usual period, indicating that the virus was not an unusual pathogenic variant. These findings support the hypothesis that the recurrent lesions seen in some human papillomavirus infections, such as recurrent laryngeal papillomatosis, are associated with specific defects in host immunity rather than variations in viral pathogenicity
Adeno-Associated Virus Type 2 Nonstructural Protein Rep78 Suppresses Translation in Vitro

AbstractAdeno-associated virus type 2 nonstructural protein Rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. In this study we examined the effects of Rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in HeLa cells and on translation of RNA encoding luciferase in rabbit reticulocyte lysate. When Rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mRNA in the cells. Purified Rep78 or Rep68 fused with Escherichia coli maltose binding protein suppressed translation of luciferase RNA in vitro, but Rep52/40 fusion proteins did not. A mutated Rep78, which is 520 aa long and truncated at its C-terminus, did suppress the in vitro translation, whereas a similarly truncated Rep78 of 420 aa did not. The results indicate that Rep78/68 function to suppress gene expression through translation inhibition, which requires the N-terminal region contained within aa 1–520
Creation and Repair of Specific DNA Double-Strand Breaks in Vivo Following Infection with Adenovirus Vectors Expressing Saccharomyces cerevisiae HO Endonuclease

AbstractTo study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to ∼30% of the available HO site genomes. Leftward fragments of ∼30 kbp, which contain the packaging signal, but not rightward fragments of ∼6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage. There was no evidence for DSB repair in E3::HO virus coinfections. In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair. To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region. The results strongly suggest that the E4 protein(s) inhibits DSB repair
Inefficient Measles Virus Budding in Murine L.CD46 Fibroblasts

AbstractInfection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L.CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L.CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation
The Cognate Coat Protein Is Required for Cell-to-Cell Movement of a Chimeric Brome Mosaic Virus Mediated by the Cucumber Mosaic Virus Movement Protein

AbstractCucumber mosaic cucumovirus (CMV) and brome mosaic bromovirus (BMV) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (CP) for cell-to-cell movement. We have shown that a chimeric BMV with the CMV 3a movement protein (MP) gene instead of its own cannot move from cell to cell in Chenopodium quinoa, a common permissive host for both BMV and CMV. Another chimeric BMV was constructed by replacing both MP and CP genes of BMV with those of CMV (MP/CP-chimera) and tested for its infectivity in C. quinoa, to determine whether the CMV CP has some functions required for the CMV MP-mediated cell-to-cell movement and to exhibit functional difference between CPs of BMV and CMV. Cell-to-cell movement of the MP/CP-chimera occurred, and small local lesions were induced on the inoculated leaves. A frameshift mutation introduced in the CMV CP gene of the MP/CP-chimera resulted in a lack of cell-to-cell movement of the chimeric virus. These results indicate that the viral movement mediated by the CMV MP requires its cognate CP. Deletion of the amino-terminal region in CMV CP, which is not obligatory for CMV movement, also abolished cell-to-cell movement of the MP/CP-chimera. This may suggest some differences in cell-to-cell movement of the MP/CP-chimera and CMV. On the other hand, the sole replacement of BMV CP gene with that of CMV abolished viral cell-to-cell movement, suggesting a possibility that the viral movement mediated by the BMV MP may also require its cognate CP. Functional compatibility between MP and CP in viral cell-to-cell movement is discussed
On the Domain Structure and the Polymerization State of the Sendai Virus P Protein

AbstractThe phosphoproteins (P) of paramyxoviruses and rhabdoviruses are cofactors of the viral polymerase (L) and chaperones of soluble nucleoprotein preventing its polymerization and nonspecific binding to cellular RNA. The primary sequences of six paramyxovirus P proteins were compared, and although there was virtually no sequence similarity, there were two regions with similar secondary structure predictions in the C-terminal part of P: the predicted multimerization domain and the X-protein, the sequence that binds to N in the N:RNA template. The C-terminal part of the Sendai virus P protein, the multimerization domain including the binding site for the polymerase, and the X-protein were expressed in Escherichia coli. All three polypeptides folded with secondary structures similar to those predicted. The C-terminal part of P is a very elongated molecule with most of its length encompassing the multimerization domain. Both the multimerization domain and the C-terminal part of P were found to form tetramers, whereas the X-protein was monomeric
Comparison of Nucleotide Sequences between Northern and Southern Philippine Isolates of Rice Grassy Stunt Virus Indicates Occurrence of Natural Genetic Reassortment

AbstractRice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5′ untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors
A Cytopathic and a Cell Culture Adapted Hepatitis A Virus Strain Differ in Cell Killing but Not in Intracellular Membrane Rearrangements

AbstractAside from a common gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype, the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Nevertheless, several cp HAV strains have been isolated during the past decade. In FRhK-4 cells infected with HM175/24a, a fast-growing cp strain, increasing amounts of viral RNA, detected by fluorescence in situ hybridization, indicated viral RNA replication. An ultrastructural analysis of the infected cells revealed a tubular–vesicular network in close proximity to the rough endoplasmic reticulum. Infection of the same cell type with a cell culture adapted (cc) strain, HM175/P35, divulged membrane alterations indistinguishable from the network induced by the cp strain. The overall appearance of the tubular–vesicular network resembles membrane alterations induced by other picornaviruses. However, the shape of the vesicle-like structures is rather oblong and tubular and, thus, seems to be specific for HAV. By electron microscopic immunocytochemistry (IEM), proteins 2B and 2C were found exclusively on the membranes of the network. Proteins expressed from the open reading frame of the cc HAV variant or 2B proteins originating from HM175 cp, cc, or the wt strain expressed in the absence of other HAV proteins induced membrane alterations resembling those seen in HAV-infected cells. The induction of similar structures suggests that protein 2B is involved in the rearrangement of cellular membranes. In all cases, IEM demonstrated that the 2B protein was closely associated with altered membranes. The extent of membrane changes did not seem to increase for both the cp strain and the cc strain during the infectious cycle. Late in the infection and shortly before the culture died off, a large number of cells infected with HM175/24a showed typical signs of apoptosis, whereas the cc strain did not induce cell killing in the same type of cells. Therefore, we conclude that cell death in HM175/24a-infected cells is induced by apoptosis rather than by cytopathology
Genetic Dissection of Tomato Bushy Stunt Virus p19-Protein-Mediated Host-Dependent Symptom Induction and Systemic Invasion

AbstractThe plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encodes a 19-kDa protein, which is translated from a 3′ proximal open reading frame (p19) that is entirely nested within the cell-to-cell movement gene (p22). Expression of the cytosolic p19-protein induces either a systemic lethal collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local lesions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determine whether these seemingly different host-dependent biological activities are governed by the same or separate regions on the 172 amino acid p19-protein. For this purpose, codons for charged amino acids predicted to be exposed on the surface of the polypeptide and presumably available for host-specific interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell movement, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. benthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutations in the central region, which substituted Arg with Gly at positions 72 or 75–78, impaired the ability of TBSV to systemically invade spinach plants. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence of protein structure effects. The implications are that regions on the N-terminal portion of the p19-protein mediate interactions in a host-dependent manner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integrity
Complementation of the Host Range Restriction of Southern Cowpea Mosaic Virus in Bean by Southern Bean Mosaic Virus

AbstractVigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea
The Cauliflower Mosaic Virus Translational Transactivator Interacts with the 60S Ribosomal Subunit Protein L18 of Arabidopsis thaliana

AbstractThe cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is involved in several aspects of the infectious cycle. P6 specifically controls the synthesis of other CaMV proteins by transactivating their expression from the polycistronic 35S RNA. By far-Western assays, we have demonstrated that P6 interacts with proteins from both healthy and CaMV-infected leaves of Arabidopsis thaliana. These proteins are found in ribosome-enriched extracts, suggesting that they participate in the translation process. One of these proteins, identified by microsequencing, corresponds to the 60S ribosomal subunit protein L18 (RPL18). Its cDNA was cloned and expressed in Escherichia coli, and the resulting RPL18 protein was shown to interact with the minimal region required for translational transactivation, designated the miniTAV domain of P6
Defining Parameters for Successful Immunocytotherapy of Persistent Viral Infection

AbstractPersistent infections with viruses such as HIV, Epstein–Barr virus, cytomelagovirus, and hepatitis B and C viruses continue to be major human health problems. Immunocytotherapy for persistent viral infections has proven successful in animal models but less effective in humans. While the requirement of antigen-specific CD8+ T cells is known, the precise role of CD4+ T cells as regards specific priming, numbers needed, and interaction with CD8+ T cells is less clear. To address these issues, we used a mouse model of persistent virus infection in which adoptive transfer of T cells effectively purges virus from all tissues. We demonstrate that (1) inclusion of antigen-specific CD4+ in addition to CD8+ T cells is mandatory for efficient and long-term virus control. Neither naive nor CD4+ T cells with specificity for a different virus are sufficient. (2) The minimal numbers of virus-specific T cells required for virus clearance from sera and tissues are 350,000 virus-specific CD8+ and 7000 virus-specific CD4+ T cells or approximately 5 × 107 CD8+ and as few as 1 × 106 CD4+ T cells per square meter of body surface area, a CD8:CD4 ratio of 50:1. (3) Production of interferon-γ, obligatory for resolution of persistent infection, is dependent on the interaction of virus-specific CD4+ and CD8+ T cells. (4) Maintenance of CD8+ T cell effector functions after adoptive transfer is directly proportional to the amount of cotransferred, virus-specific CD4+ T cells
Actin-Binding Cellular Proteins inside Human Immunodeficiency Virus Type 1

AbstractHost proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding
Replication Ability in Vitro and in Vivo of Equine Infectious Anemia Virus Avirulent Japanese Strain

AbstractAn attenuated equine infectious anemia virus (EIAV), V26, was previously prepared by 50 passages of the Japanese virulent strain V70 in primary horse macrophage culture. The horses inoculated with this V26 virus were shown to raise neutralizing antibodies against V70 without any viremia. Here, we investigated the in vitro and in vivo replication ability of V26. Comparison of the long-terminal repeat (LTR) sequences between V26 and V70 revealed a large insertion within the LTR U3 hypervariable region of V26. V26 with the mutation in the LTR showed much higher promoter activity in vitro than V70. This is consistent with the much higher replication rate of V26 in horse primary macrophage cultures compared with V70. In sharp contrast, we failed to identify the V26-specific LTR sequence by PCR, at least in sequential samples of plasma or peripheral blood mononuclear cells derived from three horses until day 62 after V26 inoculation. In contrast, antibody responses to EIAV were observed in all horses. The results suggest that the replication ability of V26 in vivo is extremely low. When one of the horses was subsequently challenged with cell-associated V70, it was found that the horse became PCR positive for EIAV. There was no LTR mutation in EIAV genome in samples periodically prepared from the V70-challenged horse. Thus it was suggested that the LTR mutation in EIAV, which occurs during serial passage in vitro, affects EIAV replication in vitro and in vivo