bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse Cytomegalovirus Immediate-Early Protein 1 Binds with Host Cell Repressors To Relieve Suppressive Effects on Viral Transcription and Replication during Lytic Infection
Authors: Tang Qiyi , Maul Gerd G.
Published: December 2002

Herpesviruses start their transcriptional cascade at nuclear domain 10 (ND10). The deposition of virus genomes at these nuclear sites occurs due to the binding of the interferon-inducible repressor protein promyelocytic leukemia protein (PML) and/or Daxx to a viral DNA-protein complex. However, the presence of repressive proteins at the nuclear site of virus transcription has remained unexplained. We investigated the mouse cytomegalovirus (MCMV) immediate-early 1 protein (IE1), which is necessary for productive infection at low multiplicities of infection and therefore likely to be involved in overcoming cellular repression. Temporal analysis of IE1 distribution revealed its initial segregation into ND10 by binding to PML and/or Daxx and IE1-dependent recruitment of the transcriptional repressor histone deacetylase-2 (HDAC-2) to this site. However, these protein aggregates are dissociated in cells producing sufficient IE1 through titration of PML, Daxx, and HDAC-2. Importantly, binding of IE1 to HDAC-2 decreased deacetylation activity. Moreover, inhibition of HDAC by trichostatin-A resulted in an increase in viral protein synthesis, an increase in cells starting the formation of prereplication compartments, and an increase in the total infectious viruses produced. Thus, IE1, like trichostatin-A, reverses the repressive effect of HDAC evident in the presence of acetylated histones in the immediate-early promoter region. Since HDAC also binds to the promoter region of IE1, as determined by the chromatin immunoprecipitation assay, these combined results suggest that IE1 inhibits or reverses HDAC-mediated repression of the infecting viral genomes, possibly by a process akin to activation of heterochromatin. We propose that even permissive cells can repress transcription of infecting viral genomes through repressors, including HDAC, Daxx, and PML, and the segregation of IE1 to ND10 that would inactivate those repressors. The virus can counter this repression by overexpressing IE1 when present in sufficient copy number, thus reducing the availability and effectiveness of these repressors.
Polymorphism and Structural Maturation of Bunyamwera Virus in Golgi and Post-Golgi Compartments
Authors: Salanueva Iñigo J. , Novoa Reyes R. , Cabezas Pilar , López-Iglesias Carmen , Carrascosa José L. , Elliott Richard M. , Risco Cristina
Published: December 2002

The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the
trans
side of the Golgi complex, reversibly blocks transformation, showing that a functional
trans
-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.
Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo
Authors: Tanaka Michiko , Kagawa Hiroyuki , Yamanashi Yuji , Sata Tetsutaro , Kawaguchi Yasushi
Published: December 2002

In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in
Escherichia coli
and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i)
E. coli
(YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by
loxP
sites was inserted into the intergenic region between U
L
3 and U
L
4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.
Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines
Authors: van 't Wout Angélique B. , Lehrman Ginger K. , Mikheeva Svetlana A. , O'Keeffe Gemma C. , Katze Michael G. , Bumgarner Roger E. , Geiss Gary K. , Mullins James I.
Published: December 2002

The expression levels of ∼4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRUinfection, consistent with the G2arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.
The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs
Authors: Ziebuhr John , Bayer Sonja , Cowley Jeff A. , Gorbalenya Alexander E.
Published: December 2002

Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus
Okavirus
, family
Roniviridae
) within the order
Nidovirales.
In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CL
pro
]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CL
pro
has a three-domain organization and is flanked by hydrophobic domains. The putative 3CL
pro
domain including flanking regions (pp1a residues 2793 to 3143) was fused to the
Escherichia coli
maltose-binding protein (MBP) and, when expressed in
E. coli
, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CL
pro
C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location
2827
LVTHE↓VRTGN
2836
. The
trans
-processing activity of the purified recombinant 3CL
pro
(pp1a residues 2832 to 3126) was used to identify another cleavage site,
6441
KVNHE↓LYHVA
6450
, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CL
pro
. The study revealed that the GAV and potyvirus 3CL
pro
s possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CL
pro
fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CL
pro
s but in common with coronavirus 3CL
pro
s, the GAV 3CL
pro
employs a Cys
2968
-His
2879
catalytic dyad. The properties of the GAV 3CL
pro
define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.
Human Immunodeficiency Virus Type 1 Nucleocapsid Zn
2+
Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA
Authors: Buckman James S. , Bosche William J. , Gorelick Robert J.
Published: December 2002

Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn
2+
finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn
2+
fingers (NC
H23C
and NC
H44C
), an integrase mutant (IN
D116N
), a double mutant (NC
H23C
/IN
D116N
), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn
2+
finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.
Two Human Immunodeficiency Virus Vaccinal Lipopeptides Follow Different Cross-Presentation Pathways in Human Dendritic Cells
Authors: Andrieu Muriel , Desoutter Jean-François , Loing Estelle , Gaston Jésintha , Hanau Daniel , Guillet Jean-Gérard , Hosmalin Anne
Published: December 2002

An efficient vaccine against human immunodeficiency virus (HIV) must induce good cellular immune responses. To do this, it must be processed and presented by dendritic cells, which are required for primary T-lymphocyte stimulation. We have previously shown that a model lipopeptide containing a short epitopic peptide from HIV-1 was endocytosed and presented in association with major histocompatibility complex class I molecules by human dendritic cells to specific CD8+T lymphocytes, but the cross-presentation pathway needed to be precisely determined. We have studied a longer lipopeptide (Pol461-484) and another lipopeptide (Nef66-97) currently being used in vaccine trials. Like the shorter lipopeptide, the rhodamine-labeled Pol461-484lipopeptide was internalized by endocytosis, as assessed by confocal microscopy. The lipopeptides were processed by dendritic cells and presented to CD8+T cells specific for the HLA-A*0201-restricted Pol476-484and the HLA-A*0301-restricted Nef73-82epitope, respectively. Presentation of both lipopeptides was inhibited by brefeldin A. Presentation of the Pol lipopeptide was inhibited by epoxomycin, a proteasome-specific inhibitor, but not by monensin. This shows that it gained access to the cytosol to be digested by the proteasome. In contrast, presentation of the Nef lipopeptide was not inhibited by epoxomycin but was inhibited by monensin, a classical inhibitor of acid-dependent endosomal enzyme activity, indicating an endocytic processing pathway yielding to major histocompatibility complex class I-restricted presentation. Therefore, the two lipopeptides followed different cross-presentation pathways, both resulting in efficient presentation to CD8+T lymphocytes.
Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum
Authors: Xu Zhenming , Choi Jinah , Lu Wen , Ou Jing-hsiung
Published: December 2002

Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.
Common Sites of Retroviral Integration in Mouse Hematopoietic Tumors Identified by High-Throughput, Single Nucleotide Polymorphism-Based Mapping and Bacterial Artificial Chromosome Hybridization
Authors: Shen Haifa , Suzuki Takeshi , Munroe David J. , Stewart Claudia , Rasmussen Lynn , Gilbert Debra J. , Jenkins Nancy A. , Copeland Neal G.
Published: December 2002

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near
Notch1
, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.
Recognition of Native Hepatitis C Virus E1E2 Heterodimers by a Human Monoclonal Antibody
Authors: Cocquerel Laurence , Quinn Elizabeth R. , Flint Mike , Hadlock Kenneth G. , Foung Steven K. H. , Levy Shoshana
Published: December 2002

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.
Baseline Susceptibility of Primary Human Immunodeficiency Virus Type 1 to Entry Inhibitors
Authors: Labrosse Béatrice , Labernardière Jean-Louis , Dam Elisabeth , Trouplin Virginie , Skrabal Katharina , Clavel François , Mammano Fabrizio
Published: December 2002

Human immunodeficiency virus type 1 plasma viruses from 29 entry inhibitor-naive patients were characterized for their susceptibilities to T-20, AMD3100, and RANTES. A strikingly wide range of susceptibilities to T-20 was observed that was influenced by coreceptor usage but not by the susceptibilities of the viruses to inhibitors that target the chemokine receptors or by polymorphisms in the gp41 N helix.
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

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

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

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

Hantaviruses 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

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

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

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

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

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

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

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

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

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

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

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.
Identification of gp120 Binding Sites on CXCR4 by Using CD4-Independent Human Immunodeficiency Virus Type 2 Env Proteins
Authors: Lin George , Baribaud Frédéric , Romano Josephine , Doms Robert W. , Hoxie James A.
Published: December 2002

Human immunodeficiency virus (HIV) and simian (SIV) immunodeficiency virus entry is mediated by binding of the viral envelope glycoprotein (Env) to CD4 and chemokine receptors, CCR5 and/or CXCR4. CD4 induces extensive conformational changes that expose and/or induce formation of a chemokine receptor binding site on gp120. CD4-independent Env's of HIV type 1 (HIV-1), HIV-2, and SIV have been identified that exhibit exposed chemokine receptor binding sites and can bind directly to CCR5 or CXCR4 in the absence of CD4. While many studies have examined determinants for gp120-CCR5 binding, analysis of gp120-CXCR4 binding has been hindered by the apparently lower affinity of this interaction for X4-tropic HIV-1 isolates. We show here that gp120 proteins from two CD4-independent HIV-2 Env's, VCP and ROD/B, bind directly to CXCR4 with an apparently high affinity. By use of CXCR4 N-terminal deletion constructs, CXCR4-CXCR2 chimeras, and human-rat CXCR4 chimeras, binding determinants were shown to reside in the amino (N) terminus, extracellular loop 2 (ECL2), and ECL3. Alanine-scanning mutagenesis of charged residues, tyrosines, and phenylalanines in extracellular CXCR4 domains implicated multiple amino acids in the N terminus (E14/E15, D20, Y21, and D22), ECL2 (D187, R188, F189, Y190, and D193), and ECL3 (D262, E268, E277, and E282) in binding, although minor differences were noted between VCP and ROD/B. However, mutations in CXCR4 that markedly reduced binding did not necessarily hinder cell-cell fusion by VCP or ROD/B, especially in the presence of CD4. These gp120 proteins will be useful in dissecting determinants for CXCR4 binding and Env triggering and in evaluating pharmacologic inhibitors of the gp120-CXCR4 interaction.
The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes
Authors: Yoshimatsu Kumiko , Lee Byoung-Hee , Araki Koichi , Morimatsu Masami , Ogino Michiko , Ebihara Hideki , Arikawa Jiro
Published: December 2002

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.
Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells
Authors: van Bleek Grada M. , Poelen Martien C. , van der Most Robbert , Brugghe Humphrey F. , Timmermans Hans A. M. , Boog Claire J. , Hoogerhout Peter , Otten Henny G. , van Els Cécile A. C. M.
Published: December 2002

Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.