bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of the Complete DNA Sequences of Human Herpesvirus 6 Variants A and B
Authors: Isegawa Yuji , Mukai Tetsu , Nakano Kazushi , Kagawa Miwa , Chen Jiguo , Mori Yasuko , Sunagawa Tomimasa , Kawanishi Kazunobu , Sashihara Junji , Hata Atsuko , Zou Ping , Kosuge Haruhiko , Yamanishi Koichi 
Published: December 2019

ABSTRACT
Human herpesvirus 6 (HHV-6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. Two variants of HHV-6 have been distinguished on the basis of differences in several properties. We have determined the complete DNA sequence of HHV-6 variant B (HHV-6B) strain HST, the causative agent of exanthem subitum, and compared the sequence with that of variant A strain U1102. A total of 115 potential open reading frames (ORFs) were identified within the 161,573-bp contiguous sequence of the entire HHV-6 genome, including some genes with remarkable differences in amino acid identity. All genes with <70% identity between the two variants were found to contain deleted regions when ORFs that could not be expressed were excluded from the comparison. Except in the case of U47, these differences were found in immediate-early/regulatory genes, DR2, DR7, U86/90, U89/90, and U95, which may represent characteristic differences of variants A and B. Also, we have successfully typed 14 different strains belonging to variant A or B by PCR using variant-specific primers; the results suggest that the remarkable differences observed were conserved evolutionarily as variant-specific divergence.
Naturally Occurring Mutations within 39 Amino Acids in the Envelope Glycoprotein of Maedi-Visna Virus Alter the Neutralization Phenotype
Authors: Skraban Robert , Matthíasdóttir Sigrídur , Torsteinsdóttir Sigurbjörg , Agnarsdóttir Gudrún , Gudmundsson Bjarki , Georgsson Gudmundur , Meloen Rob H. , Andrésson Ólafur S. , Staskus Katherine A. , Thormar Halldor , Andrésdóttir Valgerdur 
Published: December 2019

ABSTRACT
Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.
Separate Assembly and Transport Domains within the Gag Precursor of Mason-Pfizer Monkey Virus
Authors: Sakalian Michael , Hunter Eric 
Published: December 2019

ABSTRACTMason-Pfizer monkey virus (M-PMV), the prototypical type D retrovirus, assembles immature capsids within the cytoplasm of the cell prior to plasma membrane interaction. Several mutants of M-PMV Gag have been described which display altered transport, assembly, or both. In this report, we describe the use of an in vitro synthesis and assembly system to distinguish between defects in intracellular transport and the process of assembly itself for two previously describedgaggene mutants. Matrix domain mutant R55W converts the type D morphogenesis of M-PMV particles into type C and has been hypothesized to alter the transport of Gag, redirecting it to the plasma membrane where assembly subsequently occurs. We show here that R55W can assemble in both the in vitro translation-assembly system and within inclusion bodies in bacteria and thus has retained the capacity to assemble in the cytoplasm. This supports the concept that R55 is located within a domain responsible for the transport of Gag to an intracellular site for assembly. In contrast, deletions within the p12 domain of M-PMV Gag had previously been shown to affect the efficiency of particle formation such that under low-level expression conditions, Gag would fail to assemble. We demonstrate here that the efficiency of assembly in the in vitro system mirrors that seen in cells under expression conditions similar to that of an infection. These results argue that the p12 domain of this D-type retrovirus plays a critical role in the membrane-independent assembly of immature capsids.
Mutagenesis of the NS2B-NS3-Mediated Cleavage Site in the Flavivirus Capsid Protein Demonstrates a Requirement for Coordinated Processing
Authors: Amberg Sean M. , Rice Charles M. 
Published: December 2019

ABSTRACT

Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free
trans
processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a
trans
processing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation.
Human T-Cell Leukemia Virus Type 2 Rex Protein Increases Stability and Promotes Nuclear to Cytoplasmic Transport of
gag/pol
and
env
RNAs
Authors: Kusuhara Koichi , Anderson Matthew , Pettiford Sherrie M. , Green Patrick L. 
Published: December 2019

ABSTRACT

The human T-cell leukemia virus (HTLV) Rex protein is essential for efficient expression of the viral structural and enzymatic gene products. In this study, we assessed the role of the HTLV-2
rex
gene in viral RNA expression and Gag protein production. Following transfection of human JM4 T cells with wild-type and
rex
mutant full-length proviral constructs, PCR was used for semiquantitative analysis of specific viral RNA transcripts. In the presence of Rex, the total amount of steady-state viral RNA was increased fourfold. Rex significantly up-regulated the level of incompletely spliced RNAs by increasing RNA stability and was associated with a twofold down-regulation of the completely spliced
tax/rex
RNA. PCR analysis of subcellular RNA fractions, isolated from transfected cells, indicated that the level of
gag/pol
and
env
cytoplasmic RNAs were increased 7- to 9-fold in the presence of Rex, whereas Gag protein production was increased 130-fold. These data indicate that HTLV-2 Rex increases the stability and promotes nucleus-to-cytoplasm transport of the incompletely spliced viral RNAs, ultimately resulting in increased structural protein production. Moreover, this model system provides a sensitive approach to further characterize HTLV gene expression from full-length proviral clones following transfection of human T cells.
Lack of Interleukin-6 (IL-6) Enhances Susceptibility to Infection but Does Not Alter Latency or Reactivation of Herpes Simplex Virus Type 1 in IL-6 Knockout Mice
Authors: LeBlanc Rona A. , Pesnicak Lesley , Cabral Erik S. , Godleski Matthew , Straus Stephen E. 
Published: December 2019

ABSTRACTThe ability of the pleotropic, proinflammatory cytokine interleukin-6 (IL-6) to affect the replication, latency, and reactivation of herpes simplex virus type 1 (HSV-1) in cell culture and in IL-6 knockout (KO) mice was studied. In initial studies, we found no effect of exogenous IL-6, monoclonal antibodies to IL-6, or monoclonal antibody to the IL-6 coreceptor, gp130, on HSV-1 replication in vitro by plaque assay or reactivation ex vivo by explant cocultivation of latently infected murine trigeminal ganglia (TG). Compared with the wild-type (WT) mice, the IL-6 KO mice were less able to survive an ocular challenge with 105PFU of HSV-1 (McKrae) (40% survival of WT and 7% survival KO mice;P= 0.01). There was a sixfold higher 50% lethal dose of HSV-1 in WT than IL-6 KO mice (1.7 × 104and 2.7 × 103PFU, respectively). No differences were observed in titers of virus recovered from the eyes, TG, or brains or in the rates of virus reactivation by explant cocultivation of TG from latently infected WT or KO mice. Exposure of latently infected mice to UV light resulted in comparable rates of reactivation and in the proportions of WT and KO animals experiencing reactivation. Moreover, quantitative PCR assays showed nearly identical numbers of HSV-1 genomes in latently infected WT and IL-6 KO mice. These studies indicate that while IL-6 plays a role in the protection of mice from lethal HSV infection, it does not substantively influence HSV replication, spread to the nervous system, establishment of latency, or reactivation.
Regulation of Adenovirus-Mediated Transgene Expression by the Viral E4 Gene Products: Requirement for E4 ORF3
Authors: Lusky M. , Grave L. , Dieterlé A. , Dreyer D. , Christ M. , Ziller C. , Furstenberger P. , Kintz J. , Ali Hadji D. , Pavirani A. , Mehtali M. 
Published: December 2019

ABSTRACT
In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022–2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.
Protection by Live, Attenuated Simian Immunodeficiency Virus against Heterologous Challenge
Authors: Wyand Michael S. , Manson Kelledy , Montefiori David C. , Lifson Jeffrey D. , Johnson R. Paul , Desrosiers Ronald C. 
Published: December 2019

ABSTRACT

We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239Δ3, which is missing
nef
and
vpr
genes, to protect against challenge by heterologous strains SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria of CD4
+
cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of four vaccinated monkeys despite the complete mismatch of
env
sequences. However, SHIV89.6p infection was established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads were lower than those observed in unvaccinated controls but the level of protection was less than what was observed against SHIV89.6p in these experiments and considerably less than the level of protection against SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection by live, attenuated SIVmac239Δ3 against heterologous virus challenge and suggest that even live, attenuated vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation present in field strains of virus. The results further suggest that factors other than anti-Env immune responses can be principally responsible for the vaccine protection by live, attenuated SIV.
Casein Kinase 2-Mediated Phosphorylation of Respiratory Syncytial Virus Phosphoprotein P Is Essential for the Transcription Elongation Activity of the Viral Polymerase; Phosphorylation by Casein Kinase 1 Occurs Mainly at Ser
215
and Is without Effect
Authors: Dupuy Lesley C. , Dobson Sean , Bitko Vira , Barik Sailen 
Published: December 2019

ABSTRACT

The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser
232
in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser
232
was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser
215
. In vitro, P protein could be phosphorylated by purified CK1 at Ser
215
but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser
215
, Ser
232
, and Ser
237
were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.
Role of Dendritic Cells in the Immune Response Induced by Mouse Mammary Tumor Virus Superantigen
Authors: Baribaud Frédéric , Maillard Ivan , Vacheron Sonia , Brocker Thomas , Diggelmann Heidi , Acha-Orbea Hans 
Published: December 2019

ABSTRACTAfter mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4+T cells expressing Sag-specific T-cell receptor Vβ elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EαDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EαDC tg mice lacking B cells (I-EαDC tg μMT−/−), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.
Replication of GB Virus C (Hepatitis G Virus) in Interferon-Resistant Daudi Cells
Authors: Shimizu Yohko K. , Hijikata Minako , Kiyohara Tomoko , Kitamura Yoshihiro , Yoshikura Hiroshi 
Published: December 2019

ABSTRACT
We previously reported that Daudi cells, a Burkitt’s lymphoma cell line, were capable of supporting productive infection of hepatitis C virus (HCV). During continual cultivation after HCV infection, the culture became resistant to interferons (IFNs). This resistant cell line, coded as H-903, was used as host cells for replication of GB virus C (GBV-C), also known as hepatitis G virus. GBV-C RNA was detected in the culture by reverse transcription-PCR for more than 130 days after inoculation, while it was detected for 44 days but not later in the parental IFN-sensitive Daudi cells. Productive infection of GBV-C in the H-903 system was confirmed by serially inoculating supernatants from infected cultures into uninfected cells. The viral E2 antigen was detected by immunofluorescence in the cells inoculated with the fifth passage of GBV-C. The presumed capsid-coding region of the viral genome in the inoculum, in the serially passaged virus, or in the virus produced by a long-term culture was only 16 amino acids long, suggesting that the GBV-C with a short core sequence was replication competent.
Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes
Authors: Smit Jolanda M. , Bittman Robert , Wilschut Jan 
Published: December 2019

ABSTRACT
There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.