bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysticercus Antigens in Cerebrospinal Fluid Samples from Patients with Neurocysticercosis
Authors: Pardini Alessandra Xavier , Vaz Adelaide José , Machado Luis Dos Ramos , Livramento José Antônio
Published: July 2002

Antigens were detected in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis (NC) by enzyme-linked immunosorbent assay (ELISA) using polyclonal sera of rabbit anti-
Taenia solium
cysticerci (anti-Tso) and anti-
Taenia crassiceps
cysticerci vesicular fluid (anti-Tcra or anti-Tcra <30 kDa). A group of NC patients (
n
= 174) were studied (NC), including 40 patients in different phases of the disease. ELISAs carried out with the anti-Tso, anti-Tcra, and anti-Tcra <30 kDa showed sensitivities of 81.2, 90, and 95.8% and specificities of 82, 98, and 100%, respectively. The 14- and 18-kDa low-molecular-weight peptides were only detected in CSF samples from patients with NC by immunoblotting with anti-Tso and anti-Tcra sera. Because of the importance of the diagnosis and prognosis of cysticercosis, the detection of antigens may contribute as an additional marker to the study and clarification of the parasite-host relationship.
Native Valve Endocarditis Due to
Bartonella henselae
in a Middle-Aged Human Immunodeficiency Virus-Negative Woman
Authors: De La Rosa Guy R. , Barnett Ben J. , Ericsson Charles D. , Turk Jeffrey B.
Published: July 2002

We report the case of a human immunodeficiency virus-negative woman who developed native valve endocarditis of the aortic valve due to
Bartonell henselae
infection. The diagnosis was established using serology and PCR analysis of excised aortic valve tissue.
rpoB
Gene Sequence-Based Identification of
Staphylococcu
s Species
Authors: Drancourt Michel , Raoult Didier
Published: July 2002

The complete sequence of
rpoB
, the gene encoding the beta subunit of RNA polymerase was determined for
Staphylococcus saccharolyticus
,
Staphylococcus lugdunensis
, S
taphylococcus caprae
, and
Staphylococcus intermedius
and partial sequences were obtained for an additional 27
Staphylococcus
species. The complete
rpoB
sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial
rpoB
sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal
rpoB
sequence database we established enabled us to develop a molecular method for identifying
Staphylococcus
isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10
Staphylococcus
isolates, and no positive results were obtained with 10 non-
Staphylococcus
gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the
rpoB
gene as a new tool for the accurate identification of
Staphylococcus
isolates.
Poliovirus Requires a Precise 5′ End for Efficient Positive-Strand RNA Synthesis
Authors: Herold Jens , Andino Raul
Published: July 2002

Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5′ end, which are deleted during replication to generate authentic viral genomes. We removed those 5′-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5′ ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in
Xenopus laevis
oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5′ end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.
Differences in the Ability of Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) and HTLV-2 Tax To Inhibit p53 Function
Authors: Mahieux Renaud , Pise-Masison Cynthia A. , Lambert Paul F. , Nicot Christophe , De Marchis Laura , Gessain Antoine , Green Patrick , Hall William , Brady John N.
Published: July 2002

We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation.
Evolution of Circulating Wild Poliovirus and of Vaccine-Derived Poliovirus in an Immunodeficient Patient: a Unifying Model
Authors: Gavrilin Gene V. , Cherkasova Elena A. , Lipskaya Galina Y. , Kew Olen M. , Agol Vadim I.
Published: July 2002

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.
Polarization of Allogeneic T-Cell Responses by Influenza Virus-Infected Dendritic Cells
Authors: Oh Sangkon , Eichelberger Maryna C.
Published: July 2002

The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-γ) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-γ levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.
Glial Cell-Specific Regulation of the JC Virus Early Promoter by Large T Antigen
Authors: Kim Hee-Sun , Goncalves Nuno M. , Henson John W.
Published: July 2002

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease that results from an oligodendrocyte infection caused by JC virus. The JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, and thus transcriptional regulation constitutes a major mechanism of glial tropism in PML. We have previously demonstrated that T antigen controls the JC virus basal promoter in a glial cell-specific manner, since T antigen repressed the JC virus and simian virus 40 (SV40) early promoters in glioma cells but induced strong activation of the JC virus early promoter in nonglial cells. To further analyze these findings, T antigen and nuclear extracts from glial and nonglial cells were used to examine DNase I footprints on the proximal promoter. T-antigen binding to site II was more extensive than expected based on sequence homology with SV40, and nuclear proteins protected several regions of the proximal promoter in a cell-specific manner. Multiple Sp1 binding domains were identified. Site-directed mutagenesis revealed that T-antigen-mediated activation required a TATA box sequence, a pentanucleotide repeat immediately upstream of the TATA box, and an Sp1 binding site downstream of the TATA box. When footprints were obtained with mutant promoters which blocked T-antigen-induced transactivation, no change in T-antigen binding was observed. These results suggest that T antigen activates the JC virus basal promoter in nonglial cells by interaction with the transcription initiation complex.
Assembly of Double-Shelled, Virus-Like Particles in Transgenic Rice Plants Expressing Two Major Structural Proteins of Rice Dwarf Virus
Authors: Zheng Honghong , Yu Li , Wei Chunhong , Hu Dongwei , Shen Yunping , Chen Zhangliang , Li Yi
Published: July 2002

Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.
Recognition of the Core RNA Promoter for Minus-Strand RNA Synthesis by the Replicases of
Brome Mosaic Virus
and
Cucumber Mosaic Virus
Authors: Sivakumaran K. , Bao Y. , Roossinck M. J. , Kao C. C.
Published: July 2002

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the
Bromovirus
and
Cucumovirus
genera have a tRNA-like structure at the 3′ end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In
Brome mosaic virus
(BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of
Cucumber mosaic virus
(Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5′CA3′ dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.
Roles of Pr55

gag

and NCp7 in tRNA
3
Lys
Genomic Placement and the Initiation Step of Reverse Transcription in Human Immunodeficiency Virus Type 1
Authors: Cen Shan , Khorchid Ahmad , Gabor Juliana , Rong Liwei , Wainberg Mark A. , Kleiman Lawrence
Published: July 2002

To study in vivo tRNA
3
Lys
genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR
−
) virus was used as the source of primer tRNA
3
Lys
/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA
3
Lys
, dCTP, were lower with PR
−
than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR
−
dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55

gag

had no effect on initiation. These results indicate that while Pr55

gag

is sufficient for tRNA
3
Lys
placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA
3
Lys
placement and initiation of reverse transcription.
Disruption of Virion Host Shutoff Activity Improves the Immunogenicity and Protective Capacity of a Replication-Incompetent Herpes Simplex Virus Type 1 Vaccine Strain
Authors: Geiss Brian J. , Smith Tracy J. , Leib David A. , Morrison Lynda A.
Published: July 2002

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8−virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs−/ICP8−HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs−/ICP8−double mutant or vhs−or ICP8−single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs−/ICP8−mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs−virus and exceed those of mice immunized with the ICP8−single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs−/ICP8−and vhs−virus-vaccinated mice. Moreover, the double deleted vhs−/ICP8−virus protected mice better in all respects than the single deleted ICP8−mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.
Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier
Authors: Kuo Lili , Godeke Gert-Jan , Raamsman Martin J. B. , Masters Paul S. , Rottier Peter J. M.
Published: July 2002

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.
Mechanism of Assembly of Recombinant Murine Polyomavirus-Like Particles
Authors: Schmidt Uli , Rudolph Rainer , Böhm Gerald
Published: July 2002

VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro. Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid dissembly.
Involvement of both the V2 and V3 Regions of the CCR5-Tropic Human Immunodeficiency Virus Type 1 Envelope in Reduced Sensitivity to Macrophage Inflammatory Protein 1α
Authors: Maeda Yosuke , Foda Mohamed , Matsushita Shuzo , Harada Shinji
Published: July 2002

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1α-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1α (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat–β-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1β (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The
env
sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1α. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1α, MIP-1β, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.
Virus-Induced Diabetes in a Transgenic Model: Role of Cross-Reacting Viruses and Quantitation of Effector T Cells Needed To Cause Disease
Authors: Sevilla Noemi , Homann Dirk , von Herrath Matthias , Rodriguez Fernando , Harkins Stephanie , Whitton J. Lindsay , Oldstone Michael B. A.
Published: July 2002

Virus-specific cytotoxic T lymphocytes (CTL) at frequencies of >1/1,000 are sufficient to cause insulin-dependent diabetes mellitus (IDDM) in transgenic mice whose pancreatic β cells express as “self” antigen a protein from a virus later used to initiate infection. The inability to generate sufficient effector CTL for other cross-reacting viruses that fail to cause IDDM could be mapped to point mutations in the CTL epitope or its COO−flanking region. These data indicate that IDDM and likely other autoimmune diseases are caused by a quantifiable number of T cells, that neither standard epidemiologic markers nor molecular analysis with nucleic acid probes reliably distinguishes between viruses that do or do not cause diabetes, and that a single-amino-acid change flanking a CTL epitope can interfere with antigen presentation and development of autoimmune disease in vivo.
Control of Sindbis Virus Infection by Antibody in Interferon-Deficient Mice
Authors: Byrnes Andrew P. , Durbin Joan E. , Griffin Diane E.
Published: July 2002

Antibodies clear Sindbis virus from infected animals through an unknown mechanism. To determine whether interferon-induced pathways are required for this clearance, we examined mice which are unable to respond to alpha/beta interferon or gamma interferon. Although extremely susceptible to infection, such mice survived and completely cleared virus if antibodies against Sindbis virus were given.
Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors
Authors: Sarukhan Adelaida , Camugli Sabine , Gjata Bernard , von Boehmer Harald , Danos Olivier , Jooss Karin
Published: July 2002

Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4
+
T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and to achieve safe and stable gene transfer in clinical trials.
The Coronavirus Infectious Bronchitis Virus Nucleoprotein Localizes to the Nucleolus
Authors: Hiscox Julian A. , Wurm Torsten , Wilson Louise , Britton Paul , Cavanagh David , Brooks Gavin
Published: July 2002

The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.
M-X-I Motif of Semliki Forest Virus Capsid Protein Affects Nucleocapsid Assembly
Authors: Skoging-Nyberg Ulrica , Liljeström Peter
Published: July 2002

Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids.
Kinetic Analysis of the Effect of Poliovirus Receptor on Viral Uncoating: the Receptor as a Catalyst
Authors: Tsang Simon K. , McDermott Brian M. , Racaniello Vincent R. , Hogle James M.
Published: July 2002

We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.
Initiation of Minute Virus of Mice DNA Replication Is Regulated at the Level of Origin Unwinding by Atypical Protein Kinase C Phosphorylation of NS1
Authors: Nüesch Jürg P. F. , Christensen Jesper , Rommelaere Jean
Published: July 2002

Minute virus of mice nonstructural protein NS1 is a multifunctional protein that is involved in many processes necessary for virus propagation. To perform its distinct activities in timely coordinated manner, NS1 was suggested to be regulated by posttranslational modifications, in particular phosphorylation. In fact, NS1 replicative functions are dependent on protein kinase C (PKC) phosphorylation, most likely due to alteration of the biochemical profile of the viral product as determined by comparing native NS1 with its dephosphorylated counterpart. Through the characterization of NS1 mutants at individual PKC consensus phosphorylation sites for their biochemical activities and nickase function, we were able to identify two target atypical PKC phosphorylation sites, T435 and S473, serving as regulatory elements for the initiation of viral DNA replication. Furthermore, by dissociating the energy-dependent helicase activity from the ATPase-independent
trans
esterification reaction using partially single-stranded substrates, we could demonstrate that atypical PKC regulation of NS1 nickase activity occurs at the level of origin unwinding prior to
trans
esterification.
Varicella-Zoster Virus Infection of Human Dendritic Cells and Transmission to T Cells: Implications for Virus Dissemination in the Host
Authors: Abendroth Allison , Morrow Gavin , Cunningham Anthony L. , Slobedman Barry
Published: July 2002

During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% ± 6.6% (mean ± SEM) of CD1a+cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a+dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3+T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.
Mononuclear Phagocyte Differentiation, Activation, and Viral Infection Regulate Matrix Metalloproteinase Expression: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia
Authors: Ghorpade Anuja , Persidskaia Raisa , Suryadevara Radhika , Che Myhanh , Liu Xiao Juan , Persidsky Yuri , Gendelman Howard E.
Published: July 2002

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is mediated mainly by mononuclear phagocyte (MP) secretory products and their interactions with neural cells. Viral infection and MP immune activation may affect leukocyte entry into the brain. One factor that influences central nervous system (CNS) monocyte migration is matrix metalloproteinases (MMPs). In the CNS, MMPs are synthesized by resident glial cells and affect the integrity of the neuropil extracellular matrix (ECM). To ascertain how MMPs influence HAD pathogenesis, we studied their secretion following MP differentiation, viral infection, and cellular activation. HIV-1-infected and/or immune-activated monocyte-derived macrophages (MDM) and human fetal microglia were examined for production of MMP-1, -2, -3, and -9. MMP expression increased significantly with MP differentiation. Microglia secreted high levels of MMPs de novo that were further elevated following CD40 ligand-mediated cell activation. Surprisingly, HIV-1 infection of MDM led to the down-regulation of MMP-9. In encephalitic brain tissue, MMPs were expressed within perivascular and parenchymal MP, multinucleated giant cells, and microglial nodules. These data suggest that MMP production in MP is dependent on cell type, differentiation, activation, and/or viral infection. Regulation of MMP expression by these factors may contribute to neuropil ECM degradation and leukocyte migration during HAD.
RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element
Authors: Bilodeau Patricia S. , Domsic Jeffrey K. , Mayeda Akila , Krainer Adrian R. , Stoltzfus C. Martin
Published: July 2002

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two
tat
coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1
B
, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.
Hepatitis Delta Virus Minimal Substrates Competent for Editing by ADAR1 and ADAR2
Authors: Sato Shuji , Wong Swee Kee , Lazinski David W.
Published: July 2002

A host-mediated RNA-editing event allows hepatitis delta virus (HDV) to express two essential proteins, the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L), from a single open reading frame. One or several members of the ADAR (adenosine deaminases that act on RNA) family are thought to convert the adenosine to an inosine (I) within the HDAg-S amber codon in antigenomic RNA. As a consequence of replication, the UIG codon is converted to a UGG (tryptophan [W]) codon in the resulting HDAg-L message. Here, we used a novel reporter system to monitor the editing of the HDV amber/W site in the absence of replication. In cultured cells, we observed that both human ADAR1 (hADAR1) and hADAR2 were capable of editing the amber/W site with comparable efficiencies. We also defined the minimal HDV substrate required for hADAR1- and hADAR2-mediated editing. Only 24 nucleotides from the amber/W site were sufficient to enable efficient editing by hADAR1. Hence, the HDV amber/W site represents the smallest ADAR substrate yet identified. In contrast, the minimal substrate competent for hADAR2-mediated editing contained 66 nucleotides.
Tobamovirus Replicase Coding Region Is Involved in Cell-to-Cell Movement
Authors: Hirashima Kyotaro , Watanabe Yuichiro
Published: July 2002

Tobacco mosaic virus
(TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126- and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.
Specificity in Receptor Usage by T-Cell-Tropic Feline Leukemia Viruses: Implications for the In Vivo Tropism of Immunodeficiency-Inducing Variants
Authors: Lauring Adam S. , Anderson Maria M. , Overbaugh Julie
Published: July 2002

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828–1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.
Characterization of Envelope Glycoprotein Mutants for Human T-Cell Leukemia Virus Type 1 Infectivity and Immortalization
Authors: Tsukahara Tomonori , Wielgosz Matthew M. , Ratner Lee
Published: July 2002

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4+lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.
Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids
Authors: van Loo Nico-Dirk , Fortunati Elisabetta , Ehlert Erich , Rabelink Martijn , Grosveld Frank , Scholte Bob J.
Published: July 2002

We have studied the infection pathway of
Autographa californica
multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.
NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21

cip1
Authors: Op De Beeck Anne , Sobczak-Thepot Joelle , Sirma Huseyin , Bourgain Florence , Brechot Christian , Caillet-Fauquet Perrine
Published: July 2002

The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G
1
, S, and G
2
, well reproducing the arrest in S and G
2
observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21

cip1

associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21

cip1

. By comparison, MVMp infection induced the accumulation of both p53 and p21

cip1

. We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G
2
by using p53 wild-type (+/+) and null (−/−) cells. Furthermore, only the G
2
arrest was abrogated in p21

cip1

null (−/−) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21

cip1

independent, whereas the arrest in G
2
depends on both p53 and its downstream effector p21

cip1

. They also suggest that induction of p21

cip1

by the viral protein NS1 arrests cells in G
2
through inhibition of cyclin A-dependent kinase activity.
Genetic Evidence of an Essential Role for Cytomegalovirus Small Capsid Protein in Viral Growth
Authors: Borst Eva-Maria , Mathys Sibylle , Wagner Markus , Muranyi Walter , Messerle Martin
Published: July 2002

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563–7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.
Emergence in Asia of Foot-and-Mouth Disease Viruses with Altered Host Range: Characterization of Alterations in the 3A Protein
Authors: Knowles Nick J. , Davies Paul R. , Henry Tina , O'Donnell Vivian , Pacheco Juan M. , Mason Peter W.
Published: July 2002

In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.
Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis
Authors: Kolb Andreas F. , Pewe Lecia , Webster John , Perlman Stanley , Whitelaw C. Bruce A. , Siddell Stuart G.
Published: July 2002

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.