bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the 5′ Ends for Polyadenylated RNAs Synthesized during the Replication of Hepatitis Delta Virus
Authors: Gudima Severin , Dingle Kate , Wu Ting-Ting , Moraleda Gloria , Taylor John 
Published: December 2019

ABSTRACT
The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5′ rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5′ ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5′ ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5′ ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5′ ends. The observed end located at nt 1630, and most of the novel 5′ ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5′ ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5′ ends could be controlled by nucleotide sequences at and around nt 1630.
The Epstein-Barr Virus Protein BRLF1 Activates S Phase Entry through E2F1 Induction
Authors: Swenson Jennifer J. , Mauser Amy E. , Kaufmann William K. , Kenney Shannon C. 
Published: December 2019

ABSTRACT
The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545–2555, 1996).
Sendai Virus C Proteins Counteract the Interferon-Mediated Induction of an Antiviral State
Authors: Garcin Dominique , Latorre Patrizia , Kolakofsky Daniel 
Published: December 2019

ABSTRACTWe have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.
Regulation of Epstein-Barr Virus Promoters in Oral Epithelial Cells and Lymphocytes
Authors: Lagenaur Laurel A. , Palefsky Joel M. 
Published: December 2019

ABSTRACT
Hairy leukoplakia (HL) is a proliferative lesion of the tongue that supports abundant Epstein-Barr virus (EBV) replication. Previous work showed high-level expression of the EBV BMRF2 gene in HL. To characterize the regulation of BMRF2 expression in HL, we mapped the 5′ ends of the BMRF1 and BMRF2 transcripts and showed that BMRF2 is expressed from a novel internal promoter within the BMRF1 coding region. Mechanisms of BMRF2 regulation were compared in oral epithelial cells and B lymphocytes, as were those of BMRF1 and BDLF3, early and late EBV transcripts, respectively, that are also known to be expressed in HL. Basal activity of the putative BMRF2 promoter was 10-fold higher in HSC-3 epithelial cells than in B lymphocytes. The BMRF2 and the BDLF3 promoters were responsive to induction by phorbol ester, but unlike the BMRF1 promoter, they were not responsive to BZLF1 transactivation. By mutational analysis, the major activity of the BMRF2 promoter mapped to a 50-bp region, which includes a TATA-like element and a GC box. The BMRF2 promoter may be regulated differentially from the BMRF1 promoter and more closely resembles that of BDLF3. This novel BMRF2 promoter likely belongs to a class of viral promoters that is more responsive to mechanisms known to induce epithelial cell differentiation, consistent with its high level of expression in HL.
Induction of Th-1 and Th-2 Responses by Respiratory Syncytial Virus Attachment Glycoprotein Is Epitope and Major Histocompatibility Complex Independent
Authors: Srikiatkhachorn Anon , Chang Wilbur , Braciale Thomas J. 
Published: December 2019

ABSTRACTIn BALB/c mice, sensitization to respiratory syncytial virus (RSV) attachment (G) glycoprotein leads to the development of lung eosinophilia upon challenge infection with RSV, a pathology indicative of a strong in vivo induction of a Th-2-type response. In this study, we found that a strong, RSV G-specific, Th-1-type cytokine response occurred simultaneously with a Th-2-type response in G-primed mice after RSV challenge. Both Th-1 and Th-2 effector CD4+T cells recognized a single immunodominant site on this protein, implying that the differentiation of memory CD4+T cells along the Th-1 or Th-2 effector pathway was independent of the epitope specificity of the T cells. A similar observation was made in G-primedH-2bhaplotype mice after RSV challenge, further suggesting that this process is not dependent on the peptide epitope presented. On the other hand, genes mapping to loci outside of the major histocompatibility complex region are crucial regulators of the development of a Th-2-type response and lung eosinophilia. The implication of these findings for the immune mechanisms underlying the pathogenesis of RSV is discussed.
Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates
Authors: Chabot Donald J. , Zhang Peng-Fei , Quinnan Gerald V. , Broder Christopher C. 
Published: December 2019

ABSTRACTCXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4’s coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.
Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G
Authors: Feldman Steven A. , Hendry R. Michael , Beeler Judy A. 
Published: December 2019

ABSTRACT

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (
184
A→T
198
) and B (
183
K→K
197
). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence
184
A/
183
K→
198
T/K
197
for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.
Expression of Mouse Mammary Tumor Virus Superantigen mRNA in the Thymus Correlates with Kinetics of Self-Reactive T-Cell Loss
Authors: Barnett Anna , Mustafa Farah , Wrona Thomas J. , Lozano Mary , Dudley Jaquelin P. 
Published: December 2019

ABSTRACTMouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is expressed at the surface of antigen-presenting cells in conjunction with major histocompatibility complex (MHC) type II molecules. The Sag-MHC complex is recognized by entire subsets of T cells, leading to cytokine release and amplification of infected B and T cells that carry milk-borne MMTV to the mammary gland. Expression of Sag proteins from endogenous MMTV proviruses carried in the mouse germ line usually results in the deletion of self-reactive T cells during negative selection in the thymus and the elimination of T cells required for infection by specific milk-borne MMTVs. However, other endogenous MMTVs are unable to eliminate Sag-reactive T cells in newborn mice and cause partial loss of reactive T cells in adults. To investigate the kinetics of Sag-reactive T-cell deletion, backcross mice that contain single or multiple MMTVs were screened by a novel PCR assay designed to distinguish among highly related MMTV strains. Mice that containedMtv-17alone showed slow kinetics of reactive T-cell loss that involved the CD4+, but not the CD8+, subset. Deletion of CD4+or CD8+T cells reactive withMtv-17Sag was not detected in thymocytes. Slow kinetics of peripheral T-cell deletion byMtv-17Sag also was accompanied by failure to detectMtv-17 sag-specific mRNA in the thymus, despite detectable expression in other tissues, such as spleen. Together, these data suggest thatMtv-17Sag causes peripheral, rather than intrathymic, deletion of T cells. Interestingly, theMtv-8provirus caused partial deletion of CD4+Vβ12+cells in the thymus, but other T-cell subsets appeared to be deleted only in the periphery. Our data have important implications for the level of antigen expression required for elimination of self-reactive T cells. Moreover, these experiments suggest that mice expressing endogenous MMTVs that lead to slow kinetics of T-cell deletion will be susceptible to infection by milk-borne MMTVs with the same Sag specificity.
Activated Memory CD4
+
T Helper Cells Repopulate the Intestine Early following Antiretroviral Therapy of Simian Immunodeficiency Virus-Infected Rhesus Macaques but Exhibit a Decreased Potential To Produce Interleukin-2
Authors: Mattapallil Joseph J. , Smit-McBride Zeljka , Dailey Peter , Dandekar Satya 
Published: December 2019

ABSTRACT

Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we performed a longitudinal study to determine the effect of antiretroviral therapy on the phenotype and functional potential of CD4
+
T cells repopulating intestinal mucosa in human immunodeficiency virus infection. Severe depletion of CD4
+
and CD4
+
CD8
+
T cells occurred in the intestinal mucosa during primary SIV infection. The majority of these cells were of activated memory phenotype. Phosphonate 9-[2-(phosphomethoxypropyl]adenine (PMPA) treatment led to a moderate suppression of intestinal viral loads and repopulation of intestinal mucosa by predominantly activated memory CD4
+
T-helper cells. This repopulation was independent of the level of viral suppression. Compared to preinfection values, the frequency of naive CD4
+
T cells increased following PMPA therapy, suggesting that new CD4
+
T cells were repopulating the intestinal mucosa. Repopulation by CD4
+
CD8
+
T cells was not observed in either jejunum or colon lamina propria. The majority of CD4
+
T cells repopulating the intestinal mucosa following PMPA therapy were CD29
hi
and CD11a
hi
. A subset of repopulating intestinal CD4
+
T cells expressed Ki-67 antigen, indicating that local proliferation may play a role in the repopulation process. Although the majority of repopulating CD4
+
T cells in the intestinal mucosa were functionally capable of providing B- and T-cell help, as evidenced by their expression of CD28, these CD4
+
T cells were found to have a reduced capacity to produce interleukin-2 (IL-2) compared to the potential of CD4
+
T cells prior to SIV infection. Persistent viral infection may play a role in suppressing the potential of repopulating CD4
+
T cells to produce IL-2. Hence, successful antiretroviral therapy should aim at complete suppression of viral loads in mucosal lymphoid tissues, such as intestinal mucosa.
Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein
Authors: Carteau Sandrine , Gorelick Robert J. , Bushman Frederic D. 
Published: December 2019

ABSTRACT
Integration of retroviral cDNA involves coupled joining of the two ends of the viral genome at precisely spaced positions in the host cell DNA. Correct coupled joining is essential for viral replication, as shown, for example, by the finding that viral mutants defective in coupled joining are defective in integration and replication. To date, reactions with purified human immunodeficiency virus type 1 (HIV-1) integrase protein in vitro have supported mainly uncoupled joining of single cDNA ends. We have analyzed an activity stimulating coupled joining present in HIV-1 virions, which led to the finding that the HIV-1 nucleocapsid (NC) protein can stimulate coupled joining more than 1,000-fold under some conditions. The requirements for stimulating coupled joining were investigated in assays with mutant NC proteins, revealing that mutations in the zinc finger domains can influence stimulation of integration. These findings (i) provide a means for assembling more authentic integrase complexes for mechanistic studies, (ii) reveal a new activity of NC protein in vitro, (iii) indicate a possible role for NC in vivo, and (iv) provide a possible method for identifying a new class of inhibitors that disrupt coupled joining.
Variability of Human Systemic Humoral Immune Responses to Adenovirus Gene Transfer Vectors Administered to Different Organs
Authors: Harvey Ben-Gary , Hackett Neil R. , El-Sawy Tarek , Rosengart Todd K. , Hirschowitz Edward A. , Lieberman Michael D. , Lesser Martin L. , Crystal Ronald G. 
Published: December 2019

ABSTRACTAdministration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1−E3−) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 × 107to 2 × 1010particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying theEscherichia colicytosine deaminase gene) was administered (8 × 108to 8 × 109PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 × 107to 8 × 109PU, single administration) to six healthy individuals. AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 × 108to 4 × 109.5PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 102to >104. Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 × 103. Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 104. Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P> 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P= 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.
Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase
Authors: Kobasa Darwyn , Kodihalli Shantha , Luo Ming , Castrucci Maria R. , Donatelli Isabella , Suzuki Yasuo , Suzuki Takashi , Kawaoka Yoshihiro 
Published: December 2019

ABSTRACT

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between
N
-acetyl sialic acid and galactose (NeuAcα2-3Gal and NeuAcα2-6Gal) and for different molecular species of sialic acids (
N
-acetyl and
N
-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcα2-3Gal, while the activities for NeuAcα2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcα2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcα2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of
N
-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.