pinecone/core-2020-05-10-deduplication huggingface dataset:
Influenza Type B Neuraminidase Can Replace the Function of Type A Neuraminidase

AbstractInfluenza A and B viruses do not form reassortants with each other, presumably due to selection at either the RNA or protein level. Although differences in the promoter sequences of type A and B viruses have been studied, selection at the protein level has not been addressed. In this paper we describe experiments to determine whether differences in structure and/or function of the neuraminidase (NA) protein preclude formation of A/B NA reassortants. Influenza type A (N9) NA or B/Lee/40 NA expressed from plasmids can support multicycle growth of a NA-deficient type A virus (NWS-Mvi), indicating that their function in tissue culture is similar. To determine whether the type A or B NA supplied in trans can be incorporated into the virion of NWS-Mvi, the virus grown in NA-expressing cells was purified by sucrose gradient centrifugation. In each case there was a peak of NA activity coincident with the virus peak, indicating that some NA protein is packaged into the virion. The experiments suggest that, in spite of large sequence differences, the functions of the head, stalk, signal-anchor, and cytoplasmic domains of type A and B NAs are similar in tissue culture. Thus, lack of formation of A/B NA reassortant viruses is not due to restriction at the protein level
The Conserved Core of Human Immunodeficiency Virus Type 1 Nef Is Essential for Association with Lck and for Enhanced Viral Replication in T-Lymphocytes

AbstractThe Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. We report the functional interactions of the Nef proteins with Src kinases. The Nef proteins from HIV-1 and SIV bind to Lck as well as Hck, Lyn, and Fyn. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. In addition, we show that SIV and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not phosphorylate HIV-1 Nef. These data suggest that the association of Nef and Lck is central to the enhanced viral replication of HIV-1 and SIV in T-lymphocytes
Nucleocytoplasmic Export of Type D Simian Retrovirus Genomic RNA: Identification of Important Genetic Subregions and Interacting Cellular Proteins

AbstractThe simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3′ intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem–loop structure containing two major internal loops exhibiting 180° inverse symmetry, with loop face sequences A, A′, B, and B′ and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem–loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3′ IR or the subregion RNAs and that formation of RNA–protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA–protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A′, B, or B′ sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3′ two-thirds of the 3′ IR, and contain only A′ and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3′ IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA
Development of Replication-Defective Adenovirus Serotype 5 Containing the Capsid and 3C Protease Coding Regions of Foot-and-Mouth Disease Virus as a Vaccine Candidate

AbstractA recombinant replication-defective human adenovirus serotype 5 vector containing FMDV capsid, P1-2A, and viral 3C protease coding regions was constructed. Two viral clones were isolated, Ad5-P12X3CWT, containing the wild-type (WT) 3C protease that processes capsid polyprotein precursor into mature capsid proteins, and Ad5-P12X3CMUT, containing a point mutation in the protease coding region that inhibits processing. In 293 cells infected with either virus, synthesis of the FMDV capsid polyprotein precursor occurred, but processing of the polyprotein into structural proteins VP0, VP3, and VP1 occurred only in 3CWT virus-infected cells. Immunoprecipitation with monospecific and monoclonal antibodies indicates possible higher order structure formation in Ad5-P12X3CWT virus-infected cells. The viruses were used to elicit immune responses in mice inoculated intramuscularly (im). Only virus containing the 3CWT elicited a neutralizing antibody response. After boosting, this neutralizing antibody response increased. Swine inoculated im with Ad5-P12X3CWT virus developed a neutralizing antibody response and were either completely or partially protected from contact challenge with an animal directly inoculated with virulent FMDV. This adenovirus vector may be an efficient system for the delivery of FMDV cDNA into animals, leading to a high level of neutralizing antibody production and protection from FMDV challenge
Toward a Surrogate Model for Hepatitis C Virus: An Infectious Molecular Clone of the GB Virus-B Hepatitis Agent

AbstractGB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus infects tamarins, but its natural host is not known. GBV-B has special interest because it is the virus that is most closely related to hepatitis C virus (HCV), an important human pathogen. In the present study, we identified a previously unrecognized sequence at the 3′ end of the GBV-B genome. This new 3′ terminal sequence can form several predicted stem-loop structures as is typical for other members of the Flaviviridae family. We constructed molecular clones and showed that the new 3′ UTR sequence was critical for in vivo infectivity. After intrahepatic transfection of two tamarins with RNA transcripts of the full-length GBV-B clone, we detected high viral titers from Week 1 postinoculation with peak titers of ∼108 genome equivalents/ml. The viremic pattern of GBV-B infection in the transfected animals was the same as in animals inoculated intravenously with the virus pool used as the cloning source. The sequence of the recombinant virus was recovered from one of the tamarins and shown to be identical to that of the infectious clone. The development of severe hepatitis in both tamarins infected with the recombinant GBV-B virus provides formal proof that GBV-B is a true hepatitis virus
The Murine Cytomegalovirus M25 Open Reading Frame Encodes a Component of the Tegument

AbstractThe murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse–chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a λgt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643–650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument
Studies on Hybrid Comoviruses Reveal the Importance of Three-Dimensional Structure for Processing of the Viral Coat Proteins and Show That the Specificity of Cleavage Is Greater in trans Than in cis

AbstractA series of cowpea mosaic virus (CPMV)-based hybrid comoviral RNA-2 molecules have been constructed. In these, the region encoding both the large (L) and small (S) viral coat proteins was replaced by the equivalent region from bean pod mottle virus (BPMV). The hybrid RNA-2 molecules were able to replicate in cowpea protoplasts in the presence of CPMV RNA-1. Though processing of the hybrid polyproteins by the CPMV-specific 24K proteinase at the site between the 58/48K and L proteins could readily be achieved, no processing at the site between the L and S coat proteins could be obtained even when the sequence of amino acids between the two coat proteins was made CPMV-like. As a result, none of the hybrids was able to form functional virus particles, and they could not infect cowpea plants. Comparison with the processing of the L–S site in cis in reticulocyte lysates demonstrated that the requirements for processing are more stringent in trans than in cis. The results suggest that the L–S cleavage site is defined by more than just a linear sequence of amino acids and probably involves interactions between the L–S loop and the β barrels of the viral coat proteins
A Nanovirus-like DNA Component Associated with Yellow Vein Disease of Ageratum conyzoides: Evidence for Interfamilial Recombination between Plant DNA Viruses

AbstractYellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification
Aminoacylation of tRNAs Encoded by Chlorella Virus CVK2

AbstractViruses that infect certain strains of the unicellular green alga, Chlorella, have a large, linear dsDNA genome that is 330–380 kb in size; this genomic size is the largest known among viruses and is equivalent to ∼60% of the smallest prokaryotic genome of Mycoplasma genitalium (580 kb). Besides many putative protein-coding genes, a cluster of 10–15 tRNA genes is present in these viral genomes. Some of these tRNA genes contain peculiar insertions. In infected host cells, the viral tRNAs of CVK2, a Chlorella virus isolate, have been demonstrated to be cotranscribed as a large precursor, ∼1.0 kb in size, that is precisely processed into individual mature tRNA species. Acidic Northern blot analysis of eight of these tRNAs has revealed that they are actually aminoacylated in vivo, indicating their involvement in viral protein synthesis. They may help the virus reach maximal replication potential by overcoming codon usage barriers that exist between the virus and its host. These results provide evidence that some components of the host protein synthesis machinery can be replaced by viral gene products. This is the first report of tRNA aminoacylation encoded by viruses of eukaryotes
The Role of the L4 33K Gene in Adenovirus Infection

AbstractThe late phase of adenovirus infection is characterized not only by the synthesis of late proteins and the assembly of new virions, but also by the inhibition of early gene expression and host cell translation. Previous work has demonstrated that both of these inhibitory effects depend upon expression from the major late transcription unit (MLTU), controlled by the major late promoter (MLP). Furthermore, the repression of early gene expression has been shown to be mediated in trans, suggesting a role for one or more MLTU-encoded soluble factor(s). A possible candidate for such a factor is the L4-encoded 33K gene product, a protein conserved throughout the Mastadenoviridae, but of no known function. To test the role of this protein in viral infection, a stop codon was placed at the 20th position of the 33K ORF. Viable virus with genomes containing the mutation were recovered in an overlap recombination assay. Phenotypic analysis revealed that the mutant virus had a significant deficiency in both kinetics of replication and final yield, as compared to the wild-type virus. Detailed analysis of infected cells showed that there was no detectable change in the regulation of expression of several early genes and the pIX gene. This suggests either that 33K is not involved in this late phase phenomenon or that this function is replaceable by another late protein(s). Late protein synthesis and accumulation were similar to those in wild-type-infected cells. However, the reduced yield of infectious mutant virus could be accounted for by a marked deficiency in the accumulation of intermediate particles and completed capsids, suggesting a role for 33K in the process of assembly. In addition there was a small but reproducible deficiency in the shutoff of host cell translation. These results show that the 33K protein plays an important, although apparently not essential, function in the late phase of virus infection
Maintenance of Episomal SV40 Genomes in GM637 Human Fibroblasts

AbstractEpisomal SV40 (SV40: simian virus 40, Polyomavirus maccacae) has been reported in SV40-transformed human fibroblast cell lines the integrated SV40 sequences of which are unlikely to give rise to episomal copies by recombinational mechanisms. The levels of episomal viral DNA in these lines are high, being easily visualized by ethidium staining of agarose gels after electrophoresis. We find that the episomal mutant gmSV40 in GM637 cells represents a persistent lytic infection that can be cured by treatment with neutralizing antibody, leaving only the chromosomally integrated viral genomes. The finding that maintenance of the gmSV40 in GM637 cells is due to persistent infection raises a note of caution for SV40-transformed lines with episomal SV40 genomes because these lines often are used in studies of DNA replication and repair. An infective center assay that does not depend on plaque formation shows that gmSV40 is a host range mutant, with poor infectivity for CV-1 monkey kidney cells and greatly increased infectivity for human cells. Passage of gmSV40 through monkey kidney cells selects for variants with greatly increased infectivity for monkey cells and, independently, for cytopathic variants that produce plaques. Thus plaque assays can give very unreliable infective center values in studies of host range mutants
Role of Primary Constitutive Phosphorylation of Sendai Virus P and V Proteins in Viral Replication and Pathogenesis

AbstractFunctional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis
Further Requirements for Cleavage by the Murine Coronavirus 3C-like Proteinase: Identification of a Cleavage Site within ORF1b

AbstractThe coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q939↓S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL↓Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL↓Zn site, as well as the previously identified HD1↓3C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD1↓3C and POL↓Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD1↓3C site was processed more efficiently than a polypeptide substrate carrying the POL↓Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase
Release of Coronavirus E Protein in Membrane Vesicles from Virus-Infected Cells and E Protein-Expressing Cells

AbstractCoronavirus E protein is a small viral envelope protein that plays an essential role in coronavirus assembly; coexpression of coronavirus M and E proteins results in the production of virus-like particles. The present study demonstrated that mouse hepatitis virus (MHV) E protein was released as an integral membrane protein in lipid vesicles from E-protein-expressing mammalian cells, in the absence of other MHV proteins. Furthermore, our data indicated that the E-protein-containing vesicles, which had a slightly lighter buoyant density than that of MHV, were released from MHV-infected cells. These data implied that E protein alone can drive the production and release of coronavirus envelope in the absence of M protein
Loss of Dimerisation of the Nonstructural Protein NS1 of Kunjin Virus Delays Viral Replication and Reduces Virulence in Mice, but Still Allows Secretion of NS1

AbstractThe flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline250 to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine250 with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12–24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice