pinecone/core-2020-05-10-deduplication huggingface dataset:
MultipleCisElements Contribute to Geminivirus Origin Function

AbstractThe genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied intrans.These experiments demonstrated that the minimal origin is located in an 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additionalciselements—one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replicationin vivoand AL1/DNA bindingin vitro.Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multipleciselements that are likely to interact with each other during initiation of replication
The Neutralization Epitope of Lactate Dehydrogenase-Elevating Virus Is Located on the Short Ectodomain of the Primary Envelope Glycoprotein

AbstractWe have measured by indirect ELISA the binding of neutralizing and non-neutralizing anti-lactate dehydrogenase-elevating virus (LDV) polyclonal and monoclonal antibodies to synthetic peptides representing unmodified hydrophilic segments of LDV proteins. Using this method a single neutralization epitope has been shown to be located in the very short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, encoded by ORF 5. Although the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs differ slightly in amino acid sequences, the neutralizing antibodies bind strongly to the epitopes of both groups of viruses. However, the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs are associated with different numbers of polylactosaminoglycan chains (1 and 3, respectively) which may affect the binding of neutralizing antibodies to the virions of these LDVs. The ELISA using synthetic peptides containing the neutralization epitope provides a novel, rapid, sensitive, and inexpensive method for quantitating LDV neutralizing antibodies in infected mice
Intracellular Retention of Duck Hepatitis B Virus Large Surface Protein Is Independent of preS Topology

AbstractThe mechanism of intracellular retention for the large surface protein (L) of duck hepatitis B virus (DHBV) was analyzed by examination of the transmembrane topologies and secretory properties of a collection of DHBV L mutants and compared with that of human hepatitis B virus (HBV) L. Our results demonstrate that, in contrast to its HBV counterpart, intracellular retention of DHBV L does not depend on the cytosolic disposition of its preS domain: L mutants with either cytosolic or lumenal preS were mostly retained in the absence of the small surface protein (S), whereas coexpression with S resulted in efficient secretion of both topological forms. Coexpression of the wild-type DHBV L with S resulted in efficient incorporation of L into secreted S+L particles, whereas HBV L was partially excluded from secreted particles under the same conditions. We propose that HBV provides L retention even in the presence of an excess of S, by exclusion of molecules with cytosolic preS domains from secreted particles at the stage of their assembly. DHBV lacks such a retention mechanism due to the absence of topological selection in particulate assembly
Role of gp41 Glycosylation Sites in the Biological Activity of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

AbstractThe requirement for glycosylation in the transmembrane protein, gp41, of human immunodeficiency virus type 1 envelope protein for fusion activity has been studied. By using a mutant gene in which three conserved sites have been removed and which shows no fusion ability, genes were constructed which replace one, two, or three sites in all possible combinations. Following expression of the resultant proteins using the vaccinia T7 system, each Env variant was assessed by visual and quantitative syncytium assays. Our data indicate that two sites are sufficient for high levels of fusion and that the single site at position 621 is the most critical of all positions. We interpret our data in the light of previous contradictory reports on the role of gp41 glycosylation in bioactivity and the emerging structure of gp41
Characterization of the Small Subunit of the Terminase Enzyme of theBacillus subtilisBacteriophage SPP1

AbstractThe small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix–turn–helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene1of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure
A Vibriophage, KVP40, with Major Capsid Protein Homologous to gp23* of Coliphage T4

AbstractThemcpgene encoding the major capsid protein (Mcp) of vibriophage KVP40, a large-tailed DNA phage, was cloned and sequenced. The nucleotide sequence of themcpgene was 64.4% similar to that of gene23of coliphage T4. Analysis of the N-terminal amino acid sequence of purified native Mcp revealed that themcpgene actually coded for a precursor, pro-Mcp, whose 62 N-terminal amino acids must be removed upon maturation to Mcp. Thus, mature Mcp would consist of 452 amino acid residues and have a calculated molecular mass of 47,561 Da. Comparison of amino acid sequences of Mcp and gp23*, the major capsid protein of T4, demonstrated 61.8% identity and 89.7% similarity between them. In addition, a sequence, TATAAATA, identical to a typical T4 late promoter sequence was seen in the region upstream of themcpgene. These findings, together with their morphological similarity, suggest that KVP40 and T4 are phylogenetically related
DNA Requirementsin Vivofor Phage T4 Packaging

AbstractPhage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophageλimm434and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophageλimm434genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes. T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3′ region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4pacsequence. Moreover, a specific gene 16-containingpacfragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNApacsequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4
The Region of the Herpes Simplex Virus Type 1 LAT Gene Involved in Spontaneous Reactivation Does Not Encode a Functional Protein

AbstractWe previously showed that the LAT function required for efficient spontaneous reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency in the rabbit maps within the first 1.5 kb of the 8.3-kb primary LAT transcript. This demonstrated that LAT does not function via an antisense mechanism, since the first 1.5 kb of LAT does not overlap any other known HSV-1 gene. Furthermore, if LAT encodes a protein essential for efficient spontaneous reactivation, it must map within the functional first 1.5 kb of LAT. Thus, the absence of a well-conserved LAT open reading frame in this region among all HSV-1 LAT genes capable of supporting high levels of spontaneous reactivation would demonstrate that LAT does not encode a protein essential for efficient spontaneous reactivation. In this report, we sequenced the first 1.5 kb of LAT from HSV-1 McKrae, a strain with a very high spontaneous reactivation rate. Of the HSV-1 LAT sequences available for comparison (17syn+, KOS, and F), only strain 17syn+ has a high spontaneous reactivation rate. However, as shown in this report, a chimeric virus containing the KOS LAT gene on an HSV-1 McKrae genetic background had a spontaneous reactivation rate indistinguishable from McKrae (15 versus 13.6%;P> 0.05). Thus, the spontaneous reactivation competency of the LAT gene from HSV-1 KOS was similar to that of the McKrae LAT gene. Comparative sequence analysis of the LAT genes from McKrae, 17syn+, and KOS revealed that none of the eight potential McKrae LAT ORFs were well conserved. Additional types of sequence analyses further confirmed that none of the potential ORFs were likely to encode a functional LAT protein. These results strongly support the notion that the LAT function involved in spontaneous reactivation is mediated by a direct DNA or RNA mechanism rather than a protein
The Specificity of Rabies Virus RNA Encapsidation by Nucleoprotein

AbstractRabies virus nucleoprotein (N) encapsidates negative-strand genomic RNAin vivo,and this RNA–N complex, together with the nominal viral phosphoprotein (P) and RNA polymerase (L), forms the active cytoplasmic ribonucleoprotein (RNP) complex in virus-infected cells and the RNP core in virus particles. The RNP complex is capable of initiating viral RNA transcription and replicationin vivoandin vitro.To obtain insight into the events leading to the formation of the RNA–N complex, we have investigated the interaction between rabies virus N and the positive-strand leader RNA transcript. Binding studies revealed that recombinant N binds preferentially to rabies virus leader RNA and that N binding to leader RNA was 5 to 10 times stronger than to nonleader RNA. Encapsidation of leader RNA by N could be competetively inhibited by unlabeled leader RNA but not by nonleader RNA. Furthermore, N protein encapsidation of nonleader RNA but not the leader RNA was inhibited when P was simultaneously added into the encapsidation reaction, indicating that P helps confer the specificity of leader RNA encapsidation by N. The initiation signal for leader RNA encapsidation by N has been mapped to nucleotides 20–30 of the RNA sequence which is A rich. Studies with N-deletion mutants indicate that the intact N is required to encapsidate RNA, since deletion of amino acid residues from either the N- or the C-terminus of N abolishes the ability of N to encapsidate leader RNA
Intracellular Location of Two Groundnut Rosette Umbravirus Proteins Delivered by PVX and TMV Vectors

AbstractThe proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed inNicotiana benthamianaas fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions
Selective Quasispecies Transmission after Systemic or Mucosal Exposure of Macaques to Simian Immunodeficiency Virus

AbstractSexual transmission is the major cause of the AIDS epidemic. For the development of new antiviral and vaccine strategies, we therefore need to understand the mechanisms by which lentiviruses cross the mucosal barrier and the subsequent pathogenic consequences. For this purpose, experimental approaches are greatly facilitated by the development of relevant animal models. In this study, macaques were inoculated intravenously, intrarectally, or intravaginally with a pathogenic cell-free isolate of SIVmac251. Patterns of virological and immunological events significantly differed between vaginally (transient viremia, late seroconversion) and intravenously or intrarectally inoculated monkeys (persistent viremia and early seroconversion). Two weeks after infection, analysis of theenvgene nucleotide sequences of proviruses recovered from PBMCs demonstrated that most of the differences were observed in the V1 loop. Three viral variants were specifically associated with vaginal transmission, whereas no such selection was evidenced after intravenous or intrarectal transmissions. These results are in favor of specific mechanisms associated with vaginal transmission, implicating viral envelope structure
Sendai Viruses with Altered P, V, and W Protein Expression

AbstractWild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn2+-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (Δ6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8; 20–40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop= rSeV-wt ⪢ rSeV-Δ6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57Bl/6 mouse lungs and was highly virulent for these animals, whereas rSeV-Δ6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstopappeared to be more virulent for inbred C57Bl/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57Bl/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice
Arginine Residues in the C-terminus of HIV-1 Vpr Are Important for Nuclear Localization and Cell Cycle Arrest

AbstractHIV-1 viral protein R (Vpr) is predominantly localized to the nucleus and plays an important role for viral preintegration complex import into the nucleus. In this study, we investigated the influence on subcellular localization of Arg residues in the C-terminus of Vpr. Consistent with previous studies, about 90% of the cells manifested diffuse nuclear staining in the Vpr-expressed cells. Besides diffuse nuclear staining, punctate perinuclear staining, and punctate cytoplasmic staining were also observed in the immunofluorescence studies. Deletion of the Ser-Arg-Ile-Gly residues (amino acids 79–82; SRIG) had no effect on the Vpr localization. However, deletion of the Arg-Gln-Arg-Arg residues (amino acids 85–88; RQRR) resulted in a smooth perinuclear staining pattern. Substitution of five Arg residues with Asn (amino acids 80, 85, 87, 88, and 90; R → N5) resulted in a diffuse cytoplasmic staining. Subcellular fractionation analyses support the immunofluorescence staining results. These findings indicate that the C-terminal Arg residues of HIV-1 Vpr play an important role for Vpr nuclear localization. All the Vpr mutants were appropriately expressed, exhibited no significant defect on the protein stability, and were incorporated efficiently into virus-like particles. Both SRIG and R → N5 mutants lost their cell cycle arrest activities and the RQRR deletion only exhibited a low level of cell arrest activity. Therefore, the Arg residues in the HIV-1 Vpr C-terminus are important for Vpr nuclear localization and cell cycle arrest , but had no effect on protein stability or Vpr incorporation into virus-like particles
Increased Ratio ofbcl-2/baxExpression Is Associated with Bovine Leukemia Virus-Induced Leukemogenesis in Cattle

AbstractTo further investigate the molecular basis underlying the dysregulation of B cell homeostasis associated with bovine leukemia virus disease progression in cattle, bovinebaxwas cDNA cloned and sequenced. The predicted amino acid sequence of bovine Bax revealed a 192-amino-acid protein having extensive identity with the human (97%), murine (93%), and rat (94%) homologues. Because the ratio of Bcl-2 to Bax is believed to predetermine the susceptibility to a given apoptotic stimulus, the relative expression of the genes encoding these oncoproteins was evaluated in cattle naturally infected with BLV. In BLV-infected cattle an increase in the ratios ofbcl-2/baxmRNA and protein expression correlated with advancing stages of disease. These findings suggest that in addition to the maintenance of BLV-associated hematopoietic malignancies, the reciprocal expression of Bcl-2/Bax may modulate the induction of B cell expansion typical of BLV disease progression
Group I Introns Found inChlorellaViruses: Biological Implications

AbstractMore than 80 group I introns were detected and characterized inChlorellaviruses isolated from various locations in Japan; the overall average frequency of viruses containing the group I intron was 8.0%. Although most of these introns were inserted in the gene for either transcriptional elongation factor TFIIS (∼60%) or URF14.2 (unidentified open reading frame coding for a 14.2-kDa polypeptide) (∼40%), in a few cases, the gene for the major capsid protein Vp52 contained an intron. These introns were biologically active (self-splicing) bothin vivoandin vitro.Viruses that contained introns almost usually contained only one, but more than two introns coexisted in several virus isolates. Nucleotide sequence analysis showed that the intron sequences have diverged under strong constraint of the exon genes: introns in the same gene showed more than 99% sequence identity, whereas introns in different genes were only 72–78% identical. Phylogenetic analysis suggested relatedness of these introns to those found in the rRNA genes of a variety of organisms including green algae, red algae, yeasts, fungi, and protozoa
The Significance of the 3′-Nontranslated Region and E2 Amino Acid Mutations in the Virulence of Semliki Forest Virus in Mice

AbstractWe have recently shown that the 3′-nontranslated region (3′-NTR) of the avirulent Semliki Forest virus A7(74) [SFVA7(74)] contains a unique sequence of 101 nucleotides and five repetitive nucleotide units whereas the 3′-NTR of the neurovirulent SFV4 has only two repeats. A chimeric virus was constructed by replacing the entire 3′-NTR of the SFV4 clone with the A7(74) 3′-NTR. The hybrid replicated efficiently in the central nervous system (CNS) of adult Balb/c mice and, similarly to SFV4, led to high mortality after intraperitoneal inoculation. In contrast, another chimeric virus, CME2, containing the E2 gene of the avirulent SFVA7(74) virus in the SFV4 clone was recently shown to be avirulent for mice. Several derivatives with single-site or a constellation of amino acid mutations were constructed. Two single-site E2 mutants, Val37Ile and Asn212Ser, displayed an attenuated phenotype in mice with mortality reduced from 90 to 48 and 43%, respectively. None of the multiple site mutants were significantly attenuated. Adult female mice showed a greater resistance to SFV infection than male mice. The SFV hybrid viruses, CM3NTR and CME2, reached the CNS similarly to the parental viruses, but the single-site E2 mutants were only sporadically found in the CNS. We conclude that in mice the 3′-NTR does not play a significant role in the pathogenesis of Semliki Forest virus and that specific E2 amino acid mutations reduce the virulence, especially in female mice. The results additionally suggest that individual amino acid mutations in the E2 glycoprotein affect the efficiency of migration into the CNS
The Hepatitis B Virus e Antigen Cannot Pass the Murine Placenta Efficiently and Does Not Induce CTL Immune Tolerance in H-2bMicein Utero

AbstractThe function of the secretory core gene product (HBeAg) of the human hepatitis B virus is unclear. It has been discussed that this protein may be passed from the mother to the fetus, where it might induce immunologic tolerance. Here we have examined this possibility with transgenic mice expressing high levels of HBeAg. Analysis of serum samples obtained from nontrangenic fetuses which developed in HBeAg-positive mothers showed no evidence that the HBeAg can pass the placenta. Moreover, direct examination of the HBeAg- and HBcAg-specific cytotoxic T-cell immune response of H-2bmice which developed in either transgenic or nontransgenic mothers revealed no indication that mice which could have been exposed to the HBeAgin uterobecome tolerant to HBV core gene products. From these data we conclude that the placenta represents an efficient barrier for HBeAg transfer and that the HBeAg does not tolerize cytotoxic T cells, at least in mice of the H-2bhaplotype