pinecone/core-2020-05-10-deduplication huggingface dataset:
Development of a Chimeric Sindbis Virus with Enhancedper OsInfection ofAedes aegypti

AbstractThe TE/3′2J double subgenomic Sindbis (dsSIN) viruses have been used to stably express genes inAedes aegyptinerve and salivary gland tissues. However, because these viruses inefficiently infectAe. aegyptiwhen administered by theper osroute, TE/3′2J viruses must be intrathoracically inoculated into the mosquitoes to infect these tissues. A Malaysian Sindbis (SIN) virus isolate (MRE16) does efficiently infectAe. aegyptimidgut tissues after ingestion, and approximately 95% of these mosquitoes also develop disseminated infections within 14 days. We have sequenced the entire 26S RNA of MRE16 virus and have developed a chimeric SIN cDNA infectious clone, designated MRE1001, which contains sequence elements of TE/3′2J and MRE16 virus. MRE1001 virus efficiently infects midgut cells, and greater than 90% of infected mosquitoes develop disseminated infections after 14 days extrinsic incubation. The chimeric MRE1001 cDNA clone should allow identification of viral determinants of midgut infection and dissemination and lead to the development of new SIN virus expression systems
Activation of BLV Transcription by NF-κB and Tax

AbstractBovine leukemia virus (BLV) is the causative agent of bovine leukosis, a naturally occurring fatal disease in cattle. BLV transcription is regulated by cellular transcription factors and the virally encoded oncoprotein Tax. In this report, we investigated the functional role of the putative NF-κB binding site recently identified in the BLV promoter. Our studies indicate that the κB binding motif acts as a functional enhancer in the presence of the cellular NF-κB proteins. Furthermore, the κB site together with a single 21-bp repeat confers strong activation of BLV transcription in the presence the NF-κB proteins and Tax. These results suggest that cellular NF-κB may be involved in the regulation of BLV transcription and activation of the virus from latency
Atypical Splicing of the Latency-Associated Transcripts of Herpes Simplex Type 1

AbstractWe previously have shown that two latency-associated transcripts (LATs) of herpes simplex type 1 (HSV-1) are probably lariats, produced during splicing. By RNaseH digestion analysis, we now show that the major branchpoint of the 2.0-kb LAT was within 46 nt 5′ of the splice acceptor site. A more detailed mapping by primer extension revealed the branchpoint as an adenosine 29 nt 5′ of the splice acceptor site. Introduction of two branchpoint sequences with good matches to the consensus at position −25 had no effect on the splicing efficiency but reduced the accumulation of the 2.0-kb LATs at least 90-fold. The second focus of our studies was the 1.5-kb LAT. It was not detected by Northern analyses in either productively infected or transfected cultured cells or even in cells of neuronal origin. However, it was detected in the trigeminal ganglia of mice experimentally infected with HSV-1 after 10 days. Moreover, its abundance relative to that of the 2.0-kb species increased 4-fold from 10 to 30 days after infection, consistent with an interpretation that the 1.5-kb species, once formed, was more stable than the 2.0-kb species
Organization of thecis-Acting Element Required for Wheat Dwarf Geminivirus DNA Replication and Visualization of a Rep Protein–DNA Complex

AbstractInitiation of geminivirus DNA replication depends on the activity of the initiator protein (Rep) upon interaction with DNA sequences present in the intergenic region of the viral DNA. In this study, we have analyzed the DNA sequences present in the large intergenic region (LIR) of wheat dwarf virus (WDV), a subgroup I member of the geminivirus family, which are required for viral DNA replication. We have (i) defined the boundaries of the viralcis-acting DNA replication element, (ii) determined the contribution of different domains of the LIR to DNA replication efficiency, and (iii) visualized WDV Rep–DNA complexes. Analysis of unidirectional deletions from both sides of the LIR leads us to establish that a ∼200-bpcis-acting element (core) is essential for viral DNA replication. It spans ∼170 and 28 bp upstream and downstream, respectively, from the initiation site (+1), located in the invariant loop. This core element is flanked, at each side, by auxiliary regions (5′-aux and 3′-aux, ∼70 and ∼25 bp long, respectively), which contain DNA sequences that stimulate DNA replication. Competition experiments using viral replicating vectors bearing wild-type or mutant WDV LIRs suggest that the auxiliary regions may contribute to the stabilization and/or activity of the initiation complex formed by WDV Rep at the origin. We have visualized DNA–protein complexes by electron microscopy and a high-affinity binding site of WDV Rep protein within the core element has been mapped to ∼144 ± 18 bp upstream from the initiation site, between the start site for complementary-sense transcription and the TATA box. Our studies (i) establish the modular structure of the WDV DNA replicationcis-acting element and (ii) provide direct evidence for the formationin vitroof a large nucleoprotein complex within the essentialcis-acting element
Nonlinear Ribosome Migration on Cauliflower Mosaic Virus 35S RNA in Transgenic Tobacco Plants

AbstractCauliflower mosaic virus (CaMV) uses a specialised translation mechanism to bypass the long leader sequence of the 35S RNA. The effect of the CaMV 35S RNA leader sequence on the expression of a downstream β-glucuronidase (GUS) reporter gene was studied in transgenic tobacco plants. Enzymatic GUS assays of these transgenic plants show that a shunt mechanism of translation indeed occursin plantawith an average efficiency of 5% compared with the leaderless construct. Histological GUS analyses indicate that the shunt mechanism occurs throughout the whole plant and at all developmental stages
The Genomic Sequence Analysis of the Left and Right Species-Specific Terminal Region of a Cowpox Virus Strain Reveals Unique Sequences and a Cluster of Intact ORFs for Immunomodulatory and Host Range Proteins

AbstractSequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure–function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity to immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precisein vivorole(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses
Crystallization and Preliminary X-Ray Analysis of the dsDNA Bacteriophage HK97 Mature Empty Capsid

AbstractHK97 is a temperate dsDNA bacteriophage ofEscherichia colithat is structurally similar to phage lambda, with an icosahedral head of triangulation (T) number 7. Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 Å using synchrotron radiation. The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution. A capsid precursor (prohead) was madein vivoby expressing capsid proteins inE. coli.This prohead was purified, converted to Head IIin vitro,and used to grow crystals. The empty Head II has the same form as the mature HK97 capsid, but without DNA. The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling. The unit cell is monoclinic, with dimensionsa= 580 Å,b= 625 Å,c= 790 Å, β = 90.0° and two particles per unit cell
Evidence for the Separate Regulation of the Human Papillomavirus Type 11 E7 and E6 Promoters by ViralcisSequences near the E6 Promoter

AbstractThe human papillomavirus type 11 (HPV-11) E7 protein can modulate host cell functions and is required for papilloma formation, but little is known concerning the regulation of its expression. This study was designed to determine whether the viral upstream regulatory region controlled expression from the E7 promoter and whethercissequences differentially regulated E6 and E7 expression in laryngeal mucosal keratinocytes, the natural target cells for this virus. Reporter constructs were designed to study expression of the luciferase gene from the HPV-11 E7 promoter in its natural position downstream of a functional E6 promoter. E7 expression, like E6 expression, required upstream regulatory sequences. However, E7 expression was less sensitive to repression by viral E2 protein and to mutation of the SpI binding site adjacent to the E2 binding site. Moreover, there was differential sensitivity of the two promoters to mutation of the E6 TATA box, with E7 expression more affected than E6 expression. These findings show that, in the normal host cells for this virus, the E6 and E7 promoters can be independently regulated by thecisregulatory region adjacent to the E6 promoter
Nucleotide Sequence Analysis Shows thatRhopalosiphum padiVirus Is a Member of a Novel Group of Insect-Infecting RNA Viruses

AbstractRhopalosiphum padivirus (RhPV) is an aphid virus that has been considered a member of thePicornaviridaebased on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions ofDrosophilaC virus,Plautia staliintestine virus, and to a partial sequence from the 3′ end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV,DrosophilaC virus,Plautia staliintestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects
The Role of Leucine Residues in the Structure and Function of a Leucine Zipper Peptide Inhibitor of Paramyxovirus (NDV) Fusion

AbstractTo investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of synthetic peptide analogs of the sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. As previously reported (Younget al., Virology,238, 291), a peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478–497 of the F protein) inhibited syncytia formation after transfection of Cos cells with the hemagglutinin–neuraminidase and F protein cDNAs. A peptide analog which had an alanine residue in place of the first leucine residue in the zipper motif (ALDKAEESNSKLDKVNVKLT) retained inhibitory activity but less than the original peptide. Further loss in activity was observed in a peptide in which two of the leucine residues were replaced with alanine (ALDKAEESNSKADKVNVKLT), and a peptide which had all leucine residues in the zipper motif replaced with alanine (ALDKAEESNSKADKVNVKAT) had no inhibitory activity. The three-dimensional conformations of these peptides in aqueous solution were determined through the use of nuclear magnetic spectroscopy and molecular modeling. Results showed that while the wild-type peptide formed a helix with properties between an α-helix and a 310helix with leucine residues aligned along one face of the helix, progressive substitution of leucine residues with alanine resulted in the progressive loss of helical structure. The results suggest that alterations of leucine residues in the zipper motif disrupt secondary structure of the peptide and that this structure is critical to the inhibitory activity of the peptide
Feline Infectious Peritonitis Viruses Arise by Mutation from Endemic Feline Enteric Coronaviruses

AbstractFeline infectious peritonitis virus (FIPV) strains from six cats and three different geographic areas were compared genetically with feline enteric coronavirus (FECV) isolates obtained from cats inhabiting the same environments. Sequence comparisons were made from 1.2- to 8.9-kb segments on the 3′ end of the genome. FECV/FIPV pairs from the same catteries or shelters were 97.3–99.5% related but were genetically distinct from FIPV and FECV strains obtained from cats living in geographically distinct environments. The high genetic similarity between FECVs and FIPVs from the same environment strongly suggested a common ancestry. Based on the presence of deletion mutations in the FIPVs and not in the FECVs, it was concluded that FIPVs evolved as mutants of FECVs. The mutations are deletions in the FIPVs and not insertions in the FECVs since similar sequences are present in other strains that have segregated earlier from a common ancestor. Therefore, the order of descent is from FECV to FIPV. Mutations unique to FIPVs were found in open reading frames (ORFs) 3c in 4 of 6 isolates and/or 7b in 3 of 6 isolates. When the study was extended to include 7 additional FIPV isolates, 11/13 of the FIPVs sequenced were found to have mutated 3c ORFs
Demonstration of the Specificity of Poliovirus Encapsidation Using a Novel Replicon which Encodes Enzymatically Active Firefly Luciferase

AbstractThe specificity of poliovirus encapsidation has been studied using a novel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2–VP3–VP1 genes of the poliovirus capsid (P1) gene. Transfection of RNA transcribedin vitrofrom this genome resulted in a VP4-luciferase fusion protein which retained luciferase enzyme activity. Since the detection of enzyme activity was dependent upon replication of the transfected RNA genome, we refer to these genomes as replicons. The replicon encoding luciferase was encapsidated upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus, VV-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, followed by analysis of luciferase enzyme activity, revealed that encapsidated replicons could be detected at the first passage with VV-P1. Amplification of the titer of encapsidated replicons occurred upon serial passage with VV-P1, as evidenced by the high expression levels of luciferase enzyme activity following infection. Serial passage of the luciferase replicons with poliovirus type 1, 2, or 3 resulted in thetransencapsidation into the type 1, 2, or 3 capsids, respectively. In contrast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3, or enterovirus 70 did not result intransencapsidation, even though co-infection of cells with the replicon and different enteroviruses resulted in high-level expression of luciferase. The results of this study highlight the specificity of poliovirus encapsidation and point to the use of encapsidated replicons encoding luciferase as a reagent for dissecting elements of replication and encapsidation
Influence of Mucosal and Parenteral Immunization with a Replication-Defective Mutant of HSV-2 on Immune Responses and Protection from Genital Challenge

AbstractHerpes simplex virus (HSV) most frequently initiates infection at a mucosal surface; thus mucosal immune responses are likely to be important in defense against HSV infection. We have examined the effects of eliciting mucosal as well as systemic immune responses on protection against genital challenge infection with virulent HSV-2 in mice immunized with a replication-defective mutant of HSV-2. In addition, we have examined the types of immune responses elicited by immunization by the different routes under conditions known to provide protection. We observed that immunizations at parenteral and distal mucosal sites generate immune responses that have an additive effect in protection against challenge infection with virulent HSV-2. Immunization at either of these sites alone prevented paralysis and death after challenge virus infection and reduced replication of the challenge virus in the genital mucosa, although subcutaneous immunization was more effective in reducing virus replication. Simultaneous immunization at the two sites led to the greatest reduction in mucosal replication of challenge virus. The type of response generated was also affected by the route of immunization. Subcutaneous immunization results in a strong systemic immune response that is somewhat biased toward a Th1 T cell response, while intranasal immunization induces mucosal as well as systemic immunity, as evidenced by HSV-specific IgA in vaginal secretions, and a stronger bias toward a Th1 response. These results suggest that mucosal immunization may complement protective immunity against HSV-2 genital infection generated by parenteral immunization with replication-defective mutant virus
Nuclear Targeting Activity Associated with the Amino Terminal Region of the Borna Disease Virus Nucleoprotein

AbstractThe Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants lacking the 13 NH2-terminal amino acid residues1MPPKRRLVDDADA13in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of4KRR6by4NSG6was retained in the cytoplasm. Furthermore, a nonapeptide,3PKRRLVDDA11, derived from the NH2-terminal region of N conferred nuclear targeting activity to β-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where4KRR6basic amino acid residues are located
Expression of Human Papillomavirus Type 16 L1 Protein inEscherichia coli:Denaturation, Renaturation, and Self-Assembly of Virus-like Particlesin Vitro

AbstractMajor capsid protein L1 of HPV16 was produced in a fused form inEscherichia coliusing an inducible expression system. The protein formed insoluble aggregations (inclusion bodies) and the yield was more than 10% of total cell proteins. The inclusion bodies were isolated and solubilised with 8 M urea and the L1 proteins were purified by chromatographic separation. Following removal of the urea by gradual dialysis, the denatured L1 proteins spontaneously renatured and subsequently assembled into polymorphologic aggregationsin vitro.Electron microscopy showed that the assembled material included structures resembling native empty capsids as well as incompletely formed capsids. After separation from the pool of polymorphologic structures by sucrose gradient sedimentation, the correctly formed virus-like particles (VLE. coliPs) were recognised by a HPV16 type-specific, conformational-dependent monoclonal antibody in an ELISA. This system offers not only a model for investigation of the intrinsic interactions that occur during L1 assembly, but also a potential route for convenient manufacture of highly purified VLP vaccines
Function and Conformation of Wild-Type p53 Protein Are Influenced by Mutations in Bovine Leukemia Virus-Induced B-Cell Lymphosarcoma

AbstractThe mutations of the p53 gene previously represented one of several genetic changes involved in the development of bovine leukemia virus (BLV)-induced lymphosarcoma, while the effects of these mutations on the function of p53 are unknown. We identified four mutations of p53 gene in BLV-infected cattle with lymphosarcoma and demonstrated clearly the existence of two functionally distinct groups of mutants: (i) the mutant forms with substitutions at codons 241 and 242, which were mapped within an evolutionally conserved region and corresponded to the human “hot-spot” mutations, had completely lost the capacities for transactivation and growth suppression and gained transdominant repression activity in p53-null SAOS-2 cells; and (ii) the mutations at codons 206 and 207 were located outside the evolutionally conserved regions. These mutants partially retained the capacity for transactivation and growth suppression and failed to inhibit the transactivation activity of coexpressed wild-type p53, instead showing an enhancement of this activity. In addition, protein analysis using an antibody specific for the mutant form revealed that the mutations at codons 206 and 242 induced a “mutant” conformation of the bovine p53 proteins. Collectively, these results show that mutations of p53 gene in BLV-infected cattle with lymphosarcoma can potentially alter its physiological function and may play an important role in BLV-induced leukemogenesis