bluuebunny/crossref_metadata_2025_split huggingface dataset:
The High-Risk Human Papillomavirus Type 16 E6 Counters the GAP Function of E6TP1 toward Small Rap G Proteins
Authors: Singh Latika , Gao Qingshen , Kumar Ajay , Gotoh Takaya , Wazer David E. , Band Hamid , Feig Larry A. , Band Vimla 
Published: December 2002

ABSTRACT
We have recently identified E6TP1 (E6-targeted protein 1) as a novel high-risk human papillomavirus type 16 (HPV16) E6-binding protein. Importantly, mutational analysis of E6 revealed a strong correlation between the transforming activity and its abilities to bind and target E6TP1 for ubiquitin-mediated degradation. As a region within E6TP1 has high homology with GAP domains of known and putative Rap GTPase-activating proteins (GAPs), these results raised the possibility that HPV E6 may alter the Rap small-G-protein signaling pathway. Using two different approaches, we now demonstrate that human E6TP1 exhibits GAP activity for Rap1 and Rap2, confirming recent findings that a closely related rat homologue exhibits Rap-specific GAP activity. Using mutational analysis, we localize the GAP activity to residues 240 to 945 of E6TP1. Significantly, we demonstrate that coexpression of HPV16 E6, by promoting the degradation of E6TP1, enhances the GTP loading of Rap. These results support a role of Rap small-G-protein pathway in E6-mediated oncogenesis.
Amino Acid Residues 88 and 89 in the Central Hydrophilic Region of Human Immunodeficiency Virus Type 1 Vif Are Critical for Viral Infectivity by Enhancing the Steady-State Expression of Vif
Authors: Fujita Mikako , Sakurai Akiko , Yoshida Akiko , Miyaura Maki , Koyama A. Hajime , Sakai Koji , Adachi Akio 
Published: December 2002

ABSTRACT

A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E
88
WRKKR
93
) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E
88
or W
89
residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E
88
and W
89
with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E
88
or W
89
; a mutant with substitution of E
88
and W
89
with A; and a mutant with substitution of E
88
, W
89
, and R
90
with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E
88
and W
89
residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.
Transcription Initiation Activity of Adenovirus Left-End Sequence in Adenovirus Vectors with E1 Deleted
Authors: Yamamoto Masato , Davydova Julia , Takayama Koichi , Alemany Ramon , Curiel David T. 
Published: December 2002

ABSTRACT
We analyzed the transcription initiation activity of the left-end sequence (first 342 bp) of the adenovirus genome in the context of an adenovirus vector with E1 deleted in in vitro and in vivo gene transfer models. While nucleotide sequences 1 to 190 and 1 to 342 showed strong activity in three out of three lung cancer cell lines, nucleotide sequence 1 to 103 showed limited activity in H358, cells which show characteristics of type 2 alveolar cells. In vivo, the transcription initiation activities of nucleotide sequence 1 to 103 in the liver and the lung were minimal, while nucleotide sequences 1 to 190 and 1 to 342 showed strong activity comparable to that of the cytomegalovirus promoter. Further understanding of the transcription initiation activity of the left-end sequence of the adenovirus genome should lead to optimization of adenovirus vectors.
Hantavirus Pulmonary Syndrome-Associated Hantaviruses Contain Conserved and Functional ITAM Signaling Elements
Authors: Geimonen Erika , LaMonica Rachel , Springer Karen , Farooqui Yildiz , Gavrilovskaya Irina N. , Mackow Erich R. 
Published: December 2002

ABSTRACTHantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.
Yellow Fever Virus Replicons as an Expression System for Hepatitis C Virus Structural Proteins
Authors: Molenkamp Richard , Kooi Engbert A. , Lucassen Marjoleine A. , Greve Sophie , Thijssen Joyphi C. P. , Spaan Willy J. M. , Bredenbeek Peter J. 
Published: December 2002

ABSTRACT
Chimeric yellow fever virus (YF) RNAs were constructed in which the YF structural genes were replaced by the hepatitis C virus (HCV) structural genes or fusions between the YF and HCV structural genes. Interestingly, RNA replication required nucleotide complementarity between the 3′-located conserved sequence 1 and an RNA sequence located in the 5′ end of the YF capsid sequence. The (chimeric-)HCV structural proteins were efficiently expressed and processed, and the native E1/E2 heterodimer was formed. However, no indication for the production of HCV-like particles was obtained.
Valency of Antibody Binding to Enveloped Virus Particles as Determined by Surface Plasmon Resonance
Authors: Hardy Sam A. , Dimmock Nigel J. 
Published: December 2002

ABSTRACT
A simple method is described for determining the valency of binding of immunoglobulin G to immobilized influenza A virus. Where there is a free Fab arm (monovalent binding), a second virus particle is captured. This is detected by surface plasmon resonance. The methodology should be applicable to all enveloped and nonenveloped viruses.
A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3′ Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys
Authors: Whitehead Stephen S. , Falgout Barry , Hanley Kathryn A. , Blaney, Joseph E. , Markoff Lewis , Murphy Brian R. 
Published: December 2002

ABSTRACT

The Δ30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3′ untranslated region (3′ UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Δ30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Δ30 vaccine candidate. rDEN1Δ30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3′ UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Δ30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Δ30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito
Toxorhynchites splendens
. The ability of the Δ30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Δ30 mutation into wt DEN viruses belonging to each of the four serotypes.
Role of the Transmembrane Domains of prM and E Proteins in the Formation of Yellow Fever Virus Envelope
Authors: Op De Beeck Anne , Molenkamp Richard , Caron Mélanie , Ben Younes Amena , Bredenbeek Peter , Dubuisson Jean 
Published: December 2002

ABSTRACT
Flavivirus envelope proteins have been shown to play a major role in virus assembly. These proteins are anchored into cellular and viral membranes by their C-terminal domain. These domains are composed of two hydrophobic stretches separated by a short hydrophilic segment containing at least one charged residue. We investigated the role of the transmembrane domains of prM and E in the envelope formation of the flavivirus yellow fever virus (YFV). Alanine scanning insertion mutagenesis has been used to examine the role of the transmembrane domains of prM and E in YFV subviral particle formation. Most of the insertions had a dramatic effect on the release of YFV subviral particles. Some of these mutations were introduced into the viral genome. The ability of these mutant viruses to produce infectious particles was severely reduced. The alanine insertions did not affect prM-E heterodimerization. In addition, replacement of the charged residues present in the middle of the transmembrane domains had no effect on subviral particle release. Taken together, these data indicate that the transmembrane domains of prM and E play a crucial role in the biogenesis of YFV envelope. In addition, these data indicate some differences between the transmembrane domains of the hepaciviruses and the flaviviruses.
Functional Interaction of Nuclear Factor Y and Sp1 Is Required for Activation of the Epstein-Barr Virus C Promoter
Authors: Boreström Cecilia , Zetterberg Henrik , Liff Kristian , Rymo Lars 
Published: December 2002

ABSTRACT

Two Epstein-Barr virus (EBV) latent cycle promoters, Wp and Cp, are activated sequentially during virus-induced transformation of primary B lymphocytes. Immediately postinfection, viral transcription initiates from Wp, leading to expression of EBV nuclear antigen 2 (EBNA2) and EBNA5. Within 36 h, there is a switch in promoter usage from Wp to the upstream Cp, which leads to expression of EBNA1 to EBNA6. EBNA2 appears to be required for the Wp-to-Cp switch, but the switching mechanism is not fully understood at the molecular level. In a previous investigation we showed that there is an EBNA2-independent activity of reporter constructs containing deletion fragments of Cp in B-lymphoid cell lines, and we demonstrated that Cp activity is highly dependent on several cellular transcription factors, including nuclear factor Y (NF-Y) and Sp1. In the present work, we analyzed the effect of NF-Y on Cp activity in greater detail. We demonstrate that (i) a dominant negative analogue of NF-Y abolishes Cp activity, (ii) NF-Y and Sp1 costimulate Cp, and (iii) the
oriPI
-EBNA1-induced transactivation of Cp requires concomitant expression of NF-Y and Sp1, although additional factors seem necessary for optimal activation. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrate that inactivation of EBNA2 results in decreased expression of NF-Y and down-regulation of Cp. On reconstitution of the EBNA2 function, the cells enter the cell cycle, NF-Y levels increase, and a concomitant Wp-to-Cp switch occurs. Taken together, our results suggest that NF-Y is essential for Cp activation and that up-regulation of NF-Y may contribute to a successful Wp-to-Cp switch during B-cell transformation.
Conformational Changes in the Spike Glycoprotein of Murine Coronavirus Are Induced at 37°C either by Soluble Murine CEACAM1 Receptors or by pH 8
Authors: Zelus Bruce D. , Schickli Jeanne H. , Blau Dianna M. , Weiss Susan R. , Holmes Kathryn V. 
Published: December 2002

ABSTRACT

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37°C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37°C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4°C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37°C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37°C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S
A59
H716D virions with trypsin at 4°C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37°C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.
The Clearance of Hepatitis C Virus Infection in Chimpanzees May Not Necessarily Correlate with the Appearance of Acquired Immunity
Authors: Thomson Michael , Nascimbeni Michelina , Havert Michael B. , Major Marian , Gonzales Sophia , Alter Harvey , Feinstone Stephen M. , Murthy Krishna K. , Rehermann Barbara , Liang T. Jake 
Published: December 2002

ABSTRACT

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-γ)-producing CD4
+
and CD8
+
T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-γ, IFN-α, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.
Frequent Reassortment among Influenza C Viruses
Authors: Matsuzaki Y. , Mizuta K. , Sugawara K. , Tsuchiya E. , Muraki Y. , Hongo S. , Suzuki H. , Nishimura H. 
Published: December 2002

ABSTRACT

In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol.
81:
1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.
Biochemical and Genetic Studies of the VPg Uridylylation Reaction Catalyzed by the RNA Polymerase of Poliovirus
Authors: Paul Aniko V. , Peters Julia , Mugavero JoAnn , Yin Jiang , van Boom Jacques H. , Wimmer E. 
Published: December 2002

ABSTRACT

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D
pol
, UTP, and a divalent cation. The other assay uses specific viral sequences [
cre
(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD
pro
. Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D
pol
. Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D
pol
in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn
2+
as a cofactor compared to Mg
2+
or other divalent cations.
The Human Cytomegalovirus UL97 Protein Kinase, an Antiviral Drug Target, Is Required at the Stage of Nuclear Egress
Authors: Krosky Paula M. , Baek Moon-Chang , Coen Donald M. 
Published: December 2002

ABSTRACT

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a
UL97
null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.
Adenovirus Type 5 DNA Binding Protein Stimulates Binding of DNA Polymerase to the Replication Origin
Authors: van Breukelen Bas , Brenkman Arjan B. , Holthuizen P. Elly , van der Vliet Peter C. 
Published: December 2002

ABSTRACT

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the

K
m

for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.