bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Detection of Shiga Toxin-Producing Bacteria in Feces by Multiplex PCR with Molecular Beacons on the Smart Cycler
Authors: Bélanger Simon D. , Boissinot Maurice , Ménard Christian , Picard François J. , Bergeron Michel G.
Published: July 2002

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing
Escherichia coli
(STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes
stx
1
and
stx
2
, respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (
n
= 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (
n
= 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for
stx
1
and/or
stx
2
. Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.
Comparison of
gyrA
Mutations, Cyclohexane Resistance, and the Presence of Class I Integrons in
Salmonella enterica
from Farm Animals in England and Wales
Authors: Liebana Ernesto , Clouting Carol , Cassar Claire A. , Randall Luke P. , Walker Rachel A. , Threlfall E. John , Clifton-Hadley Felicity A. , Ridley Anne M. , Davies Robert H.
Published: July 2002

This study is focused on real-time detection of
gyrA
mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of
Salmonella enterica
from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (
n
= 42), followed by Ser83-Phe (
n
= 38), Ser83-Tyr (
n
= 12), Asp87-Tyr (
n
= 4), and Asp87-Gly (
n
= 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of ≥1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of ≤0.5 mg/liter. Cyclohexane resistance was more prevalent among Ser83 mutants than among Asp87 mutants (34.7 and 4%, respectively), and in 79% of isolates that presented both
gyrA
mutations and cyclohexane resistance, the level of ciprofloxacin resistance was ≥2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the
S. enterica
serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of
gyrA
mutations and of detecting class-I integrons in
Salmonella
isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.
Molecular Diagnosis of
Granulicatella elegans
on the Cardiac Valve of a Patient with Culture-Negative Endocarditis
Authors: Casalta J. P. , Habib G. , La Scola B. , Drancourt M. , Caus T. , Raoult D.
Published: July 2002

We herein report one case of culture-negative infectious endocarditis (IE) where the organism,
Granulicatella elegans
, was identified by molecular analysis using broad-range PCR primers complementary to the 16S rRNA gene on the removed valve. The results and utility of this method are discussed.
Interspecies Major Histocompatibility Complex-Restricted Th Cell Epitope on Foot-and-Mouth Disease Virus Capsid Protein VP4
Authors: Blanco Esther , McCullough Kenneth , Summerfield Artur , Fiorini Jude , Andreu David , Chiva Cristina , Borrás Eva , Barnett Paul , Sobrino Francisco
Published: July 2002

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4+CD8+cells—dominated by Th memory cells in the pig—and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.
Adenovirus Late Gene Expression Does Not Require a Rev-Like Nuclear RNA Export Pathway
Authors: Rabino Claudia , Aspegren Anders , Corbin-Lickfett Kara , Bridge Eileen
Published: July 2002

Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.
The Interaction of Vpr with Uracil DNA Glycosylase Modulates the Human Immunodeficiency Virus Type 1 In Vivo Mutation Rate
Authors: Mansky Louis M. , Preveral Sandra , Selig Luc , Benarous Richard , Benichou Serge
Published: July 2002

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to
trans
complement
vpr
null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.
Activation of Interferon Regulatory Factor 3 Is Inhibited by the Influenza A Virus NS1 Protein
Authors: Talon Julie , Horvath Curt M. , Polley Rosalind , Basler Christopher F. , Muster Thomas , Palese Peter , García-Sastre Adolfo
Published: July 2002

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-α/β) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-α/β gene expression. IRF-3 activation and, as a consequence, IFN-β mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in
trans
, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.
Lack of Functional Receptors Is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells
Authors: Mselli-Lakhal Laila , Favier Colette , Leung Kevin , Guiguen Francois , Grezel Delphine , Miossec Pierre , Mornex Jean-François , Narayan Opendra , Querat Gilles , Chebloune Yahia
Published: July 2002

Barriers to replication of viruses in potential host cells may occur at several levels. Lack of suitable and functional receptors on the host cell surface, thereby precluding entry of the virus, is a frequent reason for noninfectivity, as long as no alternative way of entry (e.g., pinocytosis, antibody-dependent adsorption) can be exploited by the virus. Other barriers can intervene at later stages of the virus life cycle, with restrictions on transcription of the viral genome, incorrect translation and posttranslational processing of viral proteins, inefficient viral assembly, and release or efficient early induction of apoptosis in the infected cell. The data we present here demonstrate that replication of caprine arthritis-encephalitis virus (CAEV) is restricted in a variety of human cell lines and primary tissue cultures. This barrier was efficiently overcome by transfection of a novel infectious complete-proviral CAEV construct into the same cells. The successful infection of human cells with a vesicular stomatitis virus (VSV) G-pseudotyped Env-defective CAEV confirmed that viral entry is the major obstacle to CAEV infection of human cells. The fully efficient productive infection obtained with the VSV-G-protein-pseudotyped infectious CAEV strengthened the evidence that lack of viral entry is the only practical barrier to CAEV replication in human cells. The virus thus produced retained its original host cell specificity and acquired no propensity to propagate further in human cultures.
Herpes Simplex Virus Types 1 and 2 Differ in Their Interaction with Heparan Sulfate
Authors: Trybala Edward , Liljeqvist Jan-Åke , Svennerholm Bo , Bergström Tomas
Published: July 2002

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC−39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.
Demyelination Determinants Map to the Spike Glycoprotein Gene of Coronavirus Mouse Hepatitis Virus
Authors: Das Sarma Jayasri , Fu Li , Tsai Jean C. , Weiss Susan R. , Lavi Ehud
Published: July 2002

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.
Isolation and Characterization of H4N6 Avian Influenza Viruses from Pigs with Pneumonia in Canada
Authors: Karasin Alexander I. , Brown Ian H. , Carman Suzanne , Olsen Christopher W.
Published: July 2002

In October 1999, H4N6 influenza A viruses were isolated from pigs with pneumonia on a commercial swine farm in Canada. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the North American lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4 influenza virus to domestic pigs under natural conditions.
Unusual Distribution of Mutations Associated with Serial Bottleneck Passages of Human Immunodeficiency Virus Type 1
Authors: Yuste Eloisa , López-Galíndez Cecilio , Domingo Esteban
Published: July 2002

Repeated bottleneck passages result in fitness losses of RNA viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations per genome, with dominance of A → G and G → A transitions (57% of all mutations) and 49% nonsynonymous replacements. One clone—but not three sibling clones—showed an overabundance of G → A transitions, evidencing the highly stochastic nature of some types of mutational bias. The distribution of mutations along the genome was very unusual in that mutation frequencies ingagwere threefold higher than inenv. Particularly striking was the complete absence of replacements in the V3 loop of gp120, confirmed with partial nucleotide sequences of additional HIV-1 clones subjected to repeated bottleneck passages. The analyses revealed several amino acid replacements that have not been previously recorded among natural HIV-1 isolates and illustrate how evolution of an RNA virus genome, with regard to constant and variable regions, can be profoundly modified by alterations in population dynamics.
Three-Dimensional Structure of the Human Herpesvirus 8 Capsid
Authors: Wu Lijun , Lo Pierrette , Yu Xuekui , Stoops James K. , Forghani B. , Zhou Z. Hong
Published: July 2002

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first description in 1994. However, its three-dimensional (3D) structure remained heretofore undetermined largely due to difficulties in viral purification. We have used log-phase cultures of body cavity-based lymphoma 1 cells induced with 12-
O
-tetradecanoylphorbol-13-acetate to obtain HHV-8 capsids for electron cryomicroscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical members of alpha- and betaherpesviruses, respectively. The inner radius of the HHV-8 capsid is identical to that of the HSV-1 capsid but is smaller than that of the HCMV capsid, which is consistent with the relative sizes of the genomes they enclose. While the HHV-8 capsid exhibits many structural similarities to the HSV-1 capsid, our reconstruction shows two major differences: its hexons lack the “horn-shaped” VP26 densities bound to the HSV-1 hexon subunits, and the HHV-8 triplexes appear smaller and less elongated than those of HSV-1. These differences are in excellent agreement with our sequence comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus capsid structure complements previous structural studies on alpha- and betaherpesviruses in providing an account of structural similarities and differences among capsids representing all human herpesvirus subfamilies.
Genetic and Biochemical Studies of Poliovirus
cis
-Acting Replication Element
cre
in Relation to VPg Uridylylation
Authors: Rieder Elizabeth , Paul Aniko V. , Kim Dong Wook , van Boom Jacques H. , Wimmer Eckard
Published: July 2002

In addition to highly conserved stem-loop structures located in the 5′- and 3′-nontranslated regions, genome replication of picornaviruses requires
cis
-acting RNA elements located in the coding region (termed
cre
) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941–1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000).
cre
elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the
cre
element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the
cre
element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D
pol
in a reaction that is greatly stimulated by 3CD
pro
(A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359–10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved
A
AACA sequence in the loop of PV-
cre
(2C) eliminated the ability of the
cre
RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; A
A
ACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AA
A
CA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-
cre
(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.
Identification and Functional Analysis of the Turnip Yellow Mosaic Tymovirus Subgenomic Promoter
Authors: Schirawski Jan , Voyatzakis Ariane , Zaccomer Bruno , Bernardi Françoise , Haenni Anne-Lise
Published: July 2002

Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.
Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion
Authors: Ignatius Ralf , Marovich Mary , Mehlhop Erin , Villamide Loreley , Mahnke Karsten , Cox William I. , Isdell Frank , Frankel Sarah S. , Mascola John R. , Steinman Ralph M. , Pope Melissa
Published: July 2002

Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.
The Herpes Simplex Virus 1 U
L
34 Protein Interacts with a Cytoplasmic Dynein Intermediate Chain and Targets Nuclear Membrane
Authors: Ye Guo-Jie , Vaughan Kevin T. , Vallee Richard B. , Roizman Bernard
Published: July 2002

To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [
35
S]methionine-labeled infected cells, two viral proteins identified as the products of U
L
34 and U
L
31 open reading frames, respectively. U
L
34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase U
S
3. U
L
31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and U
L
34 protein. In similar experiments, U
L
34 protein was found to interact with U
L
31 protein and the major capsid protein ICP5. (ii) To determine whether U
L
34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the U
L
34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed U
L
34 protein in a dose-dependent manner, and the U
L
34 protein localized primarily in the nuclear membrane. An unexpected finding was that U
L
34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. U
L
34, like many other viral proteins, may have multiple functions expressed both early and late in infection.
Role of the Influenza Virus M1 Protein in Nuclear Export of Viral Ribonucleoproteins
Authors: Bui Matthew , Wills Elizabeth G. , Helenius Ari , Whittaker Gary R.
Published: July 2002

The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117–130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.
Efficient Gene Transfer into Human CD34
+
Cells by a Retargeted Adenovirus Vector
Authors: Shayakhmetov Dmitry M. , Papayannopoulou Thalia , Stamatoyannopoulos George , Lieber André
Published: July 2002

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and α
v
integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34
+
cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34
+
cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an α
v
integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34
+
cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34
+
cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34
+
cells expressing α
v
integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34
+
cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34
+
cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34
+
c-Kit
+
cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34
+
c-Kit
+
cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.
Diminishing Returns of Population Size in the Rate of RNA Virus Adaptation
Authors: Miralles Rosario , Moya Andrés , Elena Santiago F.
Published: July 2002

Whenever an asexual viral population evolves by adapting to new environmental conditions, beneficial mutations, the ultimate cause of adaptation, are randomly produced and then fixed in the population. The larger the population size and the higher the mutation rate, the more beneficial mutations can be produced per unit time. With the usually high mutation rate of RNA viruses and in a large enough population, several beneficial mutations could arise at the same time but in different genetic backgrounds, and if the virus is asexual, they will never be brought together through recombination. Thus, the best of these genotypes must outcompete each other on their way to fixation. This competition among beneficial mutations has the effect of slowing the overall rate of adaptation. This phenomenon is known as clonal interference. Clonal interference predicts a speed limit for adaptation as the population size increases. In the present report, by varying the size of evolving vesicular stomatitis virus populations, we found evidence clearly demonstrating this speed limit and thus indicating that clonal interference might be an important factor modulating the rate of adaptation to an in vitro cell system. Several evolutionary and epidemiological implications of the clonal interference model applied to RNA viruses are discussed.
Establishment of Monoclonal Anti-Retroviral gp70 Autoantibodies from MRL/lprLupus Mice and Induction of Glomerular gp70 Deposition and Pathology by Transfer into Non-Autoimmune Mice
Authors: Tabata Nobutada , Miyazawa Masaaki , Fujisawa Ryuichi , Takei Yumiko A. , Abe Hiroyuki , Hashimoto Keiji
Published: July 2002

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutantlprgene (MRL/lprmice), F1hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70–anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lprmice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viralenvgene products. Upon transplantation, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti-gp70 autoantibodies.
Efficient Infection of Primary Tupaia Hepatocytes with Purified Human and Woolly Monkey Hepatitis B Virus
Authors: Köck Josef , Nassal Michael , MacNelly Sabine , Baumert Thomas F. , Blum Hubert E. , von Weizsäcker Fritz
Published: July 2002

The Asian tree shrew,Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.
Transfer of Hepatitis B Virus Genome by Adenovirus Vectors into Cultured Cells and Mice: Crossing the Species Barrier
Authors: Sprinzl Martin Franz , Oberwinkler Heike , Schaller Heinz , Protzer Ulrike
Published: July 2002

For the study of hepatitis B virus infection, no permissive cell line or small animal is available. Stably transfected cell lines and transgenic mice which contain hepadnavirus genomes produce virus, but—unlike in natural infection—from an integrated viral transcription template. To transfer hepadnavirus genomes across the species barrier, we developed adenovirus vectors in which 1.3-fold-overlength human and duck hepatitis B virus genomes were inserted. The adenovirus-mediated genome transfer efficiently initiated hepadnavirus replication from an extrachromosomal template in established cell lines, in primary hepatocytes from various species, and in the livers of mice. Following the transfer, hepatitis B virus proteins, genomic RNA, and all replicative DNA intermediates were detected. Detection of covalently closed circular DNA in hepatoma cell lines and in primary hepatocytes indicated that an intracellular replication cycle independent from the transferred linear viral genome was established. High-titer hepatitis B virions were released into the culture medium of hepatoma cells and the various primary hepatocytes. In addition, infectious virions were secreted into the sera of mice. In conclusion, adenovirus-mediated genome transfer initiated efficient hepatitis B virus replication in cultured liver cells and in the experimental animals from an extrachromosomal template. This will allow development of small-animal systems of hepatitis B virus infection and will facilitate study of pathogenicity of wild-type and mutant viruses as well as of virus-host interaction and new therapeutic approaches.
Autostimulation of the Epstein-Barr Virus BRLF1 Promoter Is Mediated through Consensus Sp1 and Sp3 Binding Sites
Authors: Ragoczy Tobias , Miller George
Published: July 2002

As an essential step in the lytic cascade, the Rta homologues of gammaherpesviruses all activate their own expression. Consistent with this biologic function, the Epstein-Barr virus (EBV) Rta protein powerfully stimulates the promoter of its own gene, Rp, in EBV-positive B cells in transient-transfection reporter-based assays. We analyzed the activity of RpCAT in response to Rta by deletional and site-directed mutagenesis. Two cognate Sp1 binding sites located at −279 and −45 relative to the transcriptional start site proved crucial for Rta-mediated activation. Previously described binding sites for the cellular transcription factor Zif268 and the viral transactivator ZEBRA were found to be dispensable for activation of RpCAT by Rta. Gel shift analysis, using extracts of B cells in latency or induced into the lytic cycle, identified Sp1 and Sp3 as the predominant cellular proteins bound to Rp near −45. During the lytic cycle, ZEBRA bound Rp near the Sp1/Sp3 site. The binding of Sp1 and Sp3 to Rp correlated with the reporter activities in the mutagenesis study, establishing a direct link between transcriptional activation of Rp by Rta and DNA binding by Sp1 and/or Sp3. The relative abundance or functional state of the cellular Sp1 and Sp3 transcription factors may be altered in response to stimuli that induce the BRLF1 promoter and thereby contribute to the activation of the viral lytic cycle.
Postentry Restriction to Human Immunodeficiency Virus-Based Vector Transduction in Human Monocytes
Authors: Neil Stuart , Martin Francisco , Ikeda Yasuhiro , Collins Mary
Published: July 2002

Cells of the monocyte lineage can be infected with human immunodeficiency virus type 1 (HIV-1) both during clinical infection and in vitro. The ability of HIV-1-based vectors to transduce human monocytes, monocyte-derived macrophages, and dendritic cells (DCs) was therefore examined, in order to develop an efficient protocol for antigen gene delivery to human antigen-presenting cells. Freshly isolated monocytes were refractory to HIV-1-based vector transduction but became transducible after in vitro differentiation to mature macrophages. This maturation-dependent transduction was independent of the HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef in the packaging cells and of the central polypurine tract in the vector, and it was also observed with a vesicular stomatitis virus-pseudotyped HIV-1 provirus, defective only in envelope and Nef. The level and extent of reverse transcription of the HIV-1-based vector was similar after infection of immature monocytes and of mature macrophages. However, 2LTR vector circles could not be detected in monocytes, suggesting a block to vector nuclear entry in these cells. Transduction of freshly isolated monocytes exposed to HIV-1-based vector could be rescued by subsequent differentiation into DCs. This rescue was induced by fetal calf serum in the DC culture medium, which promoted vector nuclear entry.
Herpes Simplex Virus Nucleocapsids Mature to Progeny Virions by an Envelopment → Deenvelopment → Reenvelopment Pathway
Authors: Skepper J. N. , Whiteley A. , Browne H. , Minson A.
Published: July 2002

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.
Rearrangement of the Genes of Vesicular Stomatitis Virus Eliminates Clinical Disease in the Natural Host: New Strategy for Vaccine Development
Authors: Flanagan E. Brian , Zamparo Joann M. , Ball L. Andrew , Rodriguez Luis L. , Wertz Gail W.
Published: July 2002

Gene expression among the nonsegmented negative-strand RNA viruses is controlled by distance from the single transcriptional promoter, so the phenotypes of these viruses can be systematically manipulated by gene rearrangement. We examined the potential of gene rearrangement as a means to develop live attenuated vaccine candidates againstVesicular stomatitis virus(VSV) in domestic swine, a natural host for this virus. The results showed that moving the nucleocapsid protein gene away from the single transcriptional promoter attenuated and ultimately eliminated the potential of the virus to cause disease. Combining this change with relocation of the surface glycoprotein gene yielded a vaccine that protected against challenge with wild-type VSV. By incremental manipulation of viral properties, gene rearrangement provides a new approach to generating live attenuated vaccines against this class of virus.
Microtubule Reorganization during Herpes Simplex Virus Type 1 Infection Facilitates the Nuclear Localization of VP22, a Major Virion Tegument Protein
Authors: Kotsakis Anna , Pomeranz Lisa E. , Blouin Amanda , Blaho John A.
Published: July 2002

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. (i) HSV-1(F)-induced microtubule rearrangement occurred in infected Vero cells by 13 hpi and was characterized by the loss of obvious microtubule organizing centers (MtOCs). Reformed MtOCs were detected at 25 hpi. (ii) VP22 was observed in the cytoplasm of cells prior to microtubule rearrangement and localized in the nucleus following the process. (iii) Stabilization of microtubules by the addition of taxol increased the accumulation of VP22 in the cytoplasm either during infection or in cells expressing VP22 in the absence of other viral proteins. (iv) While VP22 localized to the nuclei of cells treated with the microtubule depolymerizing agent nocodazole, either taxol or nocodazole treatment prevented optimal HSV-1(F) replication in Vero cells. (v) VP22 migration to the nucleus occurred in the presence of phosphonoacetic acid, indicating that viral DNA and true late protein synthesis were not required for its translocation. Based on these results, we conclude that (iv) microtubule reorganization during HSV-1 infection facilitates the nuclear localization of VP22.
RNA Polymerase II Holoenzyme Modifications Accompany Transcription Reprogramming in Herpes Simplex Virus Type 1-Infected Cells
Authors: Jenkins H. L. , Spencer C. A.
Published: July 2002

During lytic infection, herpes simplex virus type 1 (HSV-1) represses host transcription, recruits RNA polymerase II (RNAP II) to viral replication compartments, and alters the phosphorylation state of the RNAP II large subunit. Host transcription repression and RNAP II modifications require expression of viral immediate-early (IE) genes. Efficient modification of the RNAP II large subunit to the intermediately phosphorylated (IIi) form requires expression of ICP22 and the UL13 kinase. We have further investigated the mechanisms by which HSV-1 effects global changes in RNAP II transcription by analyzing the RNAP II holoenzyme. We find that the RNAP II general transcription factors (GTFs) remain abundant after infection and are recruited into viral replication compartments, suggesting that they continue to be involved in viral gene transcription. However, virus infection modifies the composition of the RNAP II holoenzyme, in particular triggering the loss of the essential GTF, TFIIE. Loss of TFIIE from the RNAP II holoenzyme requires viral IE gene expression, and viral IE proteins may be redundant in mediating this effect. Although viral IE proteins do not associate with the RNAP II holoenzyme, they interact with RNAP II in complexes of lower molecular mass. As the RNAP II holoenzyme containing TFIIE is necessary for activated transcription initiation and RNAP II large subunit phosphorylation in uninfected cells, virus-induced modifications to the holoenzyme may affect both of these processes, leading to aberrant phosphorylation of the RNAP II large subunit and repression of host gene transcription.
Rec (Formerly Corf) Function Requires Interaction with a Complex, Folded RNA Structure within Its Responsive Element rather than Binding to a Discrete Specific Binding Site
Authors: Magin-Lachmann Christine , Hahn Silvia , Strobel Heike , Held Ulrike , Löwer Johannes , Löwer Roswitha
Published: July 2002

It was recently reported that the human endogenous retrovirus HTDV/HERV-K encodes the regulatory protein Rec (formerly designated Corf), which is functionally equivalent to the nuclear export adapter proteins Rev of human immunodeficiency virus and Rex of human T-cell leukemia virus. We have demonstrated that the Rec protein interacts with a characteristic 429-nucleotide RNA element, the Rec-responsive element (RcRE), present in the 3′ long terminal repeat of HTDV/HERV-K transcripts. In analogy to the Rev and Rex proteins, which have distinct RNA binding sites in their responsive elements, we have proposed that Rec may also have a defined binding site in the RcRE. In this report, we demonstrate that not every HTDV/HERV-K copy present in the human genome contains an active RcRE, and we characterize mutations that abrogate Rec function. In addition, we demonstrate that Rec function requires binding to a complex, folded RNA structure rather than binding to a discrete specific binding site, in contrast to Rev and Rex and their homologous responsive elements. We define four stem-loop structures in the RcRE that are essential for Rec function. Finally, we demonstrate that both Rev and Rex can mediate nuclear export through the RcRE but that their binding sites are different from each other and from that of Rec.