bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of Interferon Action: Identification of Essential Positions within the Novel 15-Base-Pair KCS Element Required for Transcriptional Activation of the RNA-Dependent Protein Kinase
pkr
Gene
Authors: Kuhen Kelli L. , Vessey Jill W. , Samuel Charles E. 
Published: December 2019

ABSTRACT

RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (
pkr
) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5′-flanking fragments of the human
pkr
gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of the
pkr
promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5′ GGGAAGGCGGAGTCC 3′), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mouse
pkr
promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5′ NNRRRGG(
C,A,T
)GGRGYYN 3′, where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.
An Intertypic Herpes Simplex Virus Helicase-Primase Complex Associated with a Defect in Neurovirulence Has Reduced Primase Activity
Authors: Barrera I. , Bloom D. , Challberg M. 
Published: December 2019

ABSTRACT
R13-1 is an intertypic recombinant virus in which the left-hand 18% of the herpes simplex virus type 1 (HSV-1) genome is replaced by homologous sequences from HSV-2. R13-1 is nonneurovirulent and defective in DNA replication in neurons. The defect was localized to the UL5 open reading frame by using marker rescue analysis (D. C. Bloom and J. G. Stevens, J. Virol. 68:3761–3772, 1994). To provide conclusive evidence that UL5 is the only HSV-2 gene involved in the restricted replication phenotype of R13-1, we have characterized the phenotype of a recombinant virus (IB1) in which only the UL5 gene of HSV-1 was replaced by HSV-2 UL5. Data from 50% lethal dose determinations and the in vivo yields of virus suggested that IB1 has the same phenotypic characteristics as R13-1. UL5 is the helicase component of a complex with helicase and primase activities. All three subunits of this complex (UL5, UL8, and UL52) are required for viral DNA replication in all cell types. The intertypic complex HSV-2 UL5–HSV-1 UL8–HSV-1 UL52 was purified and biochemically characterized. The primase activity of the intertypic complex was 10-fold lower than that of HSV-1 UL5–HSV-1 UL8–HSV-1 UL52. The ATPase activity was comparable to that of the HSV-1 enzyme complex, and although the helicase activity was threefold lower, this did not interfere with the synthesis of leading strands by the HSV polymerase. One explanation for these findings is that the interactions between the subunits of the helicase-primase intertypic complex that are important for the full function of each subunit are inappropriate or weak.
Measles Viruses with Altered Envelope Protein Cytoplasmic Tails Gain Cell Fusion Competence
Authors: Cathomen Toni , Naim Hussein Y. , Cattaneo Roberto 
Published: December 2019

ABSTRACT
The cytoplasmic tail of the measles virus (MV) fusion (F) protein is often altered in viruses which spread through the brain of patients suffering from subacute sclerosing panencephalitis (SSPE). We transferred the coding regions of F tails from SSPE viruses in an MV genomic cDNA. Similarly, we constructed and transferred mutated tail-encoding regions of the other viral glycoprotein hemagglutinin (H) gene. From the mutated genomic cDNAs, we achieved rescue of viruses that harbor different alterations of the F tail, deletions in the membrane-distal half of the H tail, and combinations of these mutations. Viruses with alterations in any of the tails spread rapidly through the monolayer via enhanced cell-cell fusion. Double-tail mutants had even higher fusion competence but slightly decreased infectivity. Analysis of the protein composition of released mutant viral particles indicated that the tails are necessary for accurate virus envelope assembly and suggested a direct F tail-matrix (M) protein interaction. Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. We conclude that in MV infections, including SSPE, the glycoprotein tails are involved not only in virus envelope assembly but also in the control of virus-induced cell fusion.
Oral Immunization with Recombinant Norwalk Virus-Like Particles Induces a Systemic and Mucosal Immune Response in Mice
Authors: Ball Judith M. , Hardy Michele E. , Atmar Robert L. , Conner Margaret E. , Estes Mary K. 
Published: December 2019

ABSTRACTRecombinant Norwalk virus-like particles (rNV VLPs) produced in insect cells were evaluated as an oral immunogen in CD1 and BALB/c mice by monitoring rNV-specific serum total and subclass immunoglobulin G (IgG) and intestinal IgA responses. Dose and kinetics of response were evaluated in the presence and absence of the mucosal adjuvant cholera toxin (CT). rNV-specific serum IgG and intestinal IgA were detected in the absence of CT, and the number of responders was not significantly different from that of mice administered VLPs with CT at most doses. The use of CT was associated with induction of higher levels of IgG in serum; this effect was greater at higher doses of VLPs. IgG in serum was detected in the majority of animals by 9 days postimmunization (dpi), and intestinal IgA responses were detected by 24 dpi. In the absence of CT, IgG2b was the dominant IgG subclass response in both mouse strains. Thus, nonreplicating rNV VLPs are immunogenic when administered orally in the absence of any delivery system or mucosal adjuvant. These studies demonstrate that rNV VLPs are an excellent model to study the oral delivery of antigen, and they are a potential mucosal vaccine for NV infections.
Identification of Highly Attenuated Mutants of Simian Immunodeficiency Virus
Authors: Desrosiers Ronald C. , Lifson Jeffrey D. , Gibbs James S. , Czajak Susan C. , Howe Anita Y. M. , Arthur Larry O. , Johnson R. Paul 
Published: December 2019

ABSTRACT

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing
nef
,
vpr
, and upstream sequences (US) in the U3 region of the LTR (SIVmac239Δ3),
nef
,
vpx
, and US (SIVmac239Δ3x), and
nef
,
vpr
,
vpx
, and US (SIVmac239Δ4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking
vif
sequences (SIVmac239Δ
vif
) could be consistently grown only in a
vif
-complementing cell line. This Δ
vif
virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239Δ
vif
were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: Δ
vpr
> Δ
vpx
> Δ
vpr
Δ
vpx
≅ Δ
nef
> Δ3 > Δ3x ≥ Δ4 > Δ
vif
> Δ5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (Δ
vpr
and Δ
vpx
) to not detectably infectious (Δ5), simply by varying the number and location of deletions in these five loci.
Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage
Authors: André Stefanie , Seed Brian , Eberle Josef , Schraut Winfried , Bültmann Andreas , Haas Jürgen 
Published: December 2019

ABSTRACTDNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized byrev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.
Complex Formation Facilitates Endocytosis of the Varicella-Zoster Virus gE:gI Fc Receptor
Authors: Olson Julie K. , Grose Charles 
Published: December 2019

ABSTRACTOpen reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor FcγRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042–4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, ∼32%) or gI alone (steady state, ∼45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex.
Immunological and Virological Analyses of Persons Infected by Human Immunodeficiency Virus Type 1 while Participating in Trials of Recombinant gp120 Subunit Vaccines
Authors: Connor R. I. , Korber B. T. M. , Graham B. S. , Hahn B. H. , Ho D. D. , Walker B. D. , Neumann A. U. , Vermund S. H. , Mestecky J. , Jackson S. , Fenamore E. , Cao Y. , Gao F. , Kalams S. , Kunstman K. J. , McDonald D. , McWilliams N. , Trkola A. , Moore J. P. , Wolinsky S. M. 
Published: December 2019

ABSTRACTWe have studied 18 participants in phase I/II clinical trials of recombinant gp120 (rgp120) subunit vaccines (MN and SF-2) who became infected with human immunodeficiency virus type 1 (HIV-1) during the course of the trials. Of the 18 individuals, 2 had received a placebo vaccine, 9 had been immunized with MN rgp120, and seven had been immunized with SF-2 rgp120. Thirteen of the 18 infected vaccinees had received three or four immunizations prior to becoming infected. Of these, two were placebo recipients, six had received MN rgp120, and five had received SF-2 rgp120. Only 1 of the 11 rgp120 recipients who had multiple immunizations failed to develop a strong immunoglobulin G antibody response to the immunogen. However, the antibody response to rgp120 was transient, typically having a half-life of 40 to 60 days. No significant neutralizing activity against the infecting strain was detected in any of the infected individuals at any time prior to infection. Antibody titers in subjects infected despite vaccination and in noninfected subjects were not significantly different. Envelope-specific cytotoxic T-lymphocyte responses measured after infection were infrequent and weak in the nine vaccinees who were tested. HIV-1 was isolated successfully from all 18 individuals. Sixteen of these strains had a non-syncytium-inducing (NSI) phenotype, while two had a syncytium-inducing (SI) phenotype. NSI strains used the CCR5 coreceptor to enter CD4+cells, while an SI strain from one of the vaccinees also used CXCR4. Viruses isolated from the blood of rgp120 vaccinees were indistinguishable from viruses isolated from control individuals in terms of their inherent sensitivity to neutralization by specific monoclonal antibodies and their replication rates in vitro. Furthermore, genetic sequencing of theenvgenes of strains infecting the vaccinees did not reveal any features that clearly distinguished these viruses from contemporary clade B viruses circulating in the United States. Thus, despite rigorous genetic analyses, using various breakdowns of the data sets, we could find no evidence that rgp120 vaccination exerted selection pressure on the infecting HIV-1 strains. The viral burdens in the infected rgp120 vaccine recipients were also determined, and they were found to be not significantly different from those in cohorts of placebo-vaccinated and nonvaccinated individuals. In summary, we conclude that vaccination with rgp120 has had, to date, no obvious beneficial or adverse effects on the individuals we have studied.
Mutational Analysis of the Fusion Peptide of Moloney Murine Leukemia Virus Transmembrane Protein p15E
Authors: Zhu Nian-Ling , Cannon Paula M. , Chen Dagang , Anderson W. French 
Published: December 2019

ABSTRACT

Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants
trans
dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no
trans
-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.
Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization
Authors: Donzella George A. , Leon Oscar , Roth Monica J. 
Published: December 2019

ABSTRACT
Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics.
Characterization of the DNA-Binding Domain of the Avian Y-Box Protein, chkYB-2, and Mutational Analysis of Its Single-Strand Binding Motif in the Rous Sarcoma Virus Enhancer
Authors: Nambiar Ashok , Swamynathan S. K. , Kandala Jagannadha C. , Guntaka Ramareddy V. 
Published: December 2019

ABSTRACT

chkYB-2 is a sequence-specific, single-stranded DNA binding chicken Y-box protein that promotes Rous sarcoma virus long terminal repeat (RSV LTR)-driven transcription in avian fibroblasts. The DNA-binding domain of chkYB-2 has been mapped by characterizing the DNA binding properties of purified recombinant chkYB-2 mutant polypeptides. The data indicate that the invariant cold shock domain (CSD) is necessary but not sufficient for association with DNA and suggest that another conserved region, adjacent to the carboxyl boundary of the CSD, plays a role in high-affinity DNA binding. chkYB-2 binds to a tandem repeat of the 5′-GTACCACC-3′ motif on the RSV LTR. Mutational analysis of this recognition sequence revealed the requirement of an essentially unaltered template for both high-affinity binding by chkYB-2 as well as maximal transcriptional activity of the RSV LTR in vivo. The single-stranded DNA binding activity of chkYB-2 is augmented by Mg
2+
. The possible significance of this finding for transactivation by a single-strand DNA binding protein is discussed.
Nitric Oxide Inhibits Rhinovirus-Induced Cytokine Production and Viral Replication in a Human Respiratory Epithelial Cell Line
Authors: Sanders Scherer P. , Siekierski Edward S. , Porter Jacqueline D. , Richards Stephen M. , Proud David 
Published: December 2019

ABSTRACTTo better understand the early biochemical events that occur in human rhinovirus (HRV) infections, we examined the kinetics and mechanisms of interleukin-8 (IL-8) and IL-6 production from infected epithelial cells. Several HRV strains caused IL-8 and IL-6 production, but HRV-16 induced maximal IL-8 and IL-6 mRNA expression and protein production more rapidly than did HRV-14, despite similar rates of replication of the two viral strains. Viral induction of cytokine mRNA does not require new protein synthesis, since it was unaffected by cycloheximide treatment. The potent glucocorticoid budesonide did not affect viral replication or cytokine mRNA induction but modestly inhibited cytokine protein production. Interestingly, the nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (NONOate) inhibited both rhinovirus replication and cytokine production in a dose-dependent fashion without reducing levels of cytokine mRNA. The NONOate effects were due to release of nitric oxide, because NONOate that had been depleted of its nitric oxide content had no effect. Thus, nitric oxide may play an important anti-inflammatory and antiviral role in colds and nitric oxide donors may represent a novel therapeutic approach.
DNA Immunization against Herpes Simplex Virus: Enhanced Efficacy Using a Sindbis Virus-Based Vector
Authors: Hariharan Mangala J. , Driver David A. , Townsend Kay , Brumm Duane , Polo John M. , Belli Barbara A. , Catton Donald J. , Hsu David , Mittelstaedt Denise , McCormack James E. , Karavodin Linda , Dubensky Thomas W. , Chang Stephen M. W. , Banks Theresa A. 
Published: December 2019

ABSTRACTPreviously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508–519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.
Mapping Viral DNA Specificity to the Central Region of Integrase by Using Functional Human Immunodeficiency Virus Type 1/Visna Virus Chimeric Proteins
Authors: Katzman Michael , Sudol Malgorzata 
Published: December 2019

ABSTRACT
We previously described the construction and analysis of the first set of functional chimeric lentivirus integrases, involving exchange of the N-terminal, central, and C-terminal regions of the human immunodeficiency virus type 1 (HIV-1) and visna virus integrase (IN) proteins. Based on those results, additional HIV-1/visna virus chimeric integrases were designed and purified. Each of the chimeric enzymes was functional in at least one oligonucleotide-based IN assay. Of a total of 12 chimeric IN proteins, 3 exhibit specific viral DNA processing, 9 catalyze insertion of viral DNA ends, 12 can reverse that reaction, and 11 are active for nonspecific alcoholysis. Functional data obtained with the processing assay indicate that the central region of the protein is responsible for viral DNA specificity. Target site selection for nonspecific alcoholysis again mapped to the central domain of IN, confirming our previous data indicating that this region can position nonviral DNA for nucleophilic attack. However, the chimeric proteins created patterns of viral DNA insertion distinct from that of either wild-type IN, suggesting that interactions between regions of IN influence target site selection for viral DNA integration. The results support a new model for the functional organization of IN in which viral DNA initially binds nonspecifically to the C-terminal portion of IN but the catalytic central region of the enzyme has a prominent role both in specific recognition of viral DNA ends and in positioning the host DNA for viral DNA integration.