bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal Ribosomal Entry Site-Mediated Translation Initiation in Equine Rhinitis A Virus: Similarities to and Differences from That of Foot-and-Mouth Disease Virus
Authors: Hinton Tracey M. , Li Feng , Crabb Brendan S.
Published: July 2002

Equine rhinitis A virus
(ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of
Foot-and-mouth disease virus
(FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5′ nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.
Cap-Independent Translational Enhancement of Turnip Crinkle Virus Genomic and Subgenomic RNAs
Authors: Qu Feng , Morris T. Jack
Published: July 2002

The presence of translational control elements and cap structures has not been carefully investigated for members of the
Carmovirus
genus, a group of small icosahedral plant viruses with positive-sense RNA genomes. In this study, we examined both the 5′ and 3′ untranslated regions (UTRs) of the turnip crinkle carmovirus (TCV) genomic RNA (4 kb) as well as the 5′ UTR of the coat protein subgenomic RNA (1.45 kb) for their roles in translational regulation. All three UTRs enhanced translation of the firefly luciferase reporter gene to different extents. Optimal translational efficiency was achieved when mRNAs contained both 5′ and 3′ UTRs. The synergistic effect due to the 5′-3′ cooperation was at least fourfold greater than the sum of the contributions of the individual UTRs. The observed translational enhancement of TCV mRNAs occurred in a cap-independent manner, a result consistent with the demonstration, using a cap-specific antibody, that the 5′ end of the TCV genomic RNA was uncapped. Finally, the translational enhancement activity within the 5′ UTR of 1.45-kb subgenomic RNA was shown to be important for the translation of coat protein in protoplasts and for virulent infection in
Arabidopsis
plants.
Possible Involvement of the Double-Stranded RNA-Binding Core Protein ςA in the Resistance of Avian Reovirus to Interferon
Authors: Martínez-Costas José , González-López Claudia , Vakharia Vikram N. , Benavente Javier
Published: July 2002

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein ςA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein ςA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.
Cytokine Expression, Natural Killer Cell Activation, and Phenotypic Changes in Lymphoid Cells from Rhesus Macaques during Acute Infection with Pathogenic Simian Immunodeficiency Virus
Authors: Giavedoni Luis D. , Velasquillo M. Cristina , Parodi Laura M. , Hubbard Gene B. , Hodara Vida L.
Published: July 2002

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3−CD16+lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4+T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4+T cells and absent in peripheral blood leukocyte (PBL) CD4+T cells, was down-regulated in LN CD4+T cells and up-regulated in PBL CD4+T cells immediately after infection. CD8+T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4+T cells but not in LN CD4+T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection.
Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein
Authors: Anderson Mark G. , Scoggin Kirsten E. S. , Simbulan-Rosenthal Cynthia M. , Steadman Jennifer A.
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax.
Characterization of Vaccinia Virus Intracellular Cores: Implications for Viral Uncoating and Core Structure
Authors: Pedersen Ketil , Snijder Eric J. , Schleich Sibylle , Roos Norbert , Griffiths Gareth , Locker Jacomine Krijnse
Published: July 2002

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40–dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.
Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus
Authors: Magden Julia , Takeda Naokazu , Li Tiancheng , Auvinen Petri , Ahola Tero , Miyamura Tatsuo , Merits Andres , Kääriäinen Leevi
Published: July 2002

Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group fromS-adenosylmethionine (AdoMet) to GTP and GDP to yield m7GTP or m7GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m7GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m7GMP, in the presence of AdoMet and GTP, because radioactivity from both [α-32P]GTP and [3H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m7GTP, m7GDP, et2m7GMP, and m2et7GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.
Molecular Basis for the Interaction of the Hepatitis B Virus Core Antigen with the Surface Immunoglobulin Receptor on Naive B Cells
Authors: Lazdina Una , Cao Tinghua , Steinbergs Juris , Alheim Mats , Pumpens Paul , Peterson Darrel L. , Milich David R. , Leroux-Roels Geert , Sällberg Matti
Published: July 2002

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLκV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4+T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.
Cellular Changes Induced by Low-Risk Human Papillomavirus Type 11 in Keratinocytes That Stably Maintain Viral Episomes
Authors: Thomas Jennifer T. , Oh Stephen T. , Terhune Scott S. , Laimins Laimonis A.
Published: July 2002

Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174–4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.
PA Subunit from Influenza Virus Polymerase Complex Interacts with a Cellular Protein with Homology to a Family of Transcriptional Activators
Authors: Huarte Maite , Sanz-Ezquerro Juan Jose , Roncal Fernando , Ortı́n Juan , Nieto Amelia
Published: July 2002

The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from
Gallus gallus
called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.
Latency-Associated Nuclear Antigen Encoded by Kaposi's Sarcoma-Associated Herpesvirus Interacts with Tat and Activates the Long Terminal Repeat of Human Immunodeficiency Virus Type 1 in Human Cells
Authors: Hyun Teresa S. , Subramanian Chitra , Cotter Murray A. , Thomas Robert A. , Robertson Erle S.
Published: July 2002

The latency-associated nuclear antigen (LANA) is constitutively expressed in cells infected with the Kaposi's sarcoma (KS) herpesvirus (KSHV), also referred to as human herpesvirus 8. KSHV is tightly associated with body cavity-based lymphomas (BCBLs) in immunocompromised patients infected with human immunodeficiency virus (HIV). LANA, encoded by open reading frame 73 of KSHV, is one of a small subset of proteins expressed during latent infection and was shown to be important in tethering the viral episome to host chromosomes. Additionally, it has been shown that LANA can function as a regulator of transcription. However, its role in the progression of disease is still being elucidated. Since KS is one of the most common AIDS-associated cancers in the United States and BCBLs appear predominantly in AIDS patients, we examined whether LANA is able to regulate the HIV type 1 (HIV-1) long terminal repeat (LTR). Using luciferase-based transient transfection assays, we found that LANA was able to transactivate the HIV-1 LTR in the human B-cell line BJAB, human monocytic cell line U937, and the human embryonic kidney fibroblast cell line 293T. Moreover, we observed that the virus-encoded HIV transactivator protein Tat cooperated with LANA in activation of the LTR in a dose-response fashion with increasing amounts of LANA. Surprisingly, LANA alone was sufficient to transactivate the HIV-1 LTR in BJAB cells. In similar assays using a HIV-1 LTR construct with the core enhancer elements deleted; the activity of LANA was diminished but not abolished, indicating a mechanism which involves the cooperation of the core enhancer elements and downstream elements which include Tat. Furthermore, transient transfection of an infectious clone of HIV with LANA demonstrated effects similar to those seen in the reporter assays based on Western blot analysis of HIV Gag polypeptide p24. Interestingly, we also demonstrated that the carboxy terminus of LANA associates with Tat in cells and in vitro. These experiments suggest a role for LANA in activating the HIV-1 LTR through association with cellular molecules targeting the core enhancer elements and Tat and may have important consequences in increasing the levels of HIV in infected individuals and, hence, the disease state.
Recombinant Expression of Lymphocytic Choriomeningitis Virus Strain WE Glycoproteins: a Single Amino Acid Makes the Difference
Authors: Beyer Winfried R. , Miletic Hrvoje , Ostertag Wolfram , von Laer Dorothee
Published: July 2002

Cytoplasmic vector systems are generally used for expression of lymphocytic choriomeningitis virus (LCMV) proteins. However, we achieved high levels of cell surface glycoproteins using a standard nuclear expression plasmid. Expression was independent of other LCMV proteins but was blocked by a missense mutation within the original LCMV(WE) glycoprotein cDNA.
In Situ Spatial Organization of Potato Virus A Coat Protein Subunits as Assessed by Tritium Bombardment
Authors: Baratova Ludmila A. , Efimov Aleksander V. , Dobrov Eugenie N. , Fedorova Natalija V. , Hunt Reet , Badun Gennadii A. , Ksenofontov Aleksander L. , Torrance Lesley , Järvekülg Lilian
Published: July 2002

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing α-helices and β-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two β-strands, (ii) a C-terminal region including two α-helices, as well as three β-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four α-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.
CD4 Down-Modulation by Human Immunodeficiency Virus Type 1 Nef Correlates with the Efficiency of Viral Replication and with CD4
+
T-Cell Depletion in Human Lymphoid Tissue Ex Vivo
Authors: Glushakova Svetlana , Münch Jan , Carl Silke , Greenough Thomas C. , Sullivan John L. , Margolis Leonid , Kirchhoff Frank
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo
.
We analyzed recombinant and primary
nef
alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4
+
T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4
+
T cells in lymphoid tissues and accelerate AIDS progression.
Structural Analysis of the −1 Ribosomal Frameshift Elements in Giardiavirus mRNA
Authors: Li Lei , Wang Alice L. , Wang Ching C.
Published: July 2002

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a −1 ribosomal frameshift in a region where
gag
and
pol
open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5′ 631-nt cDNA and the out-of-frame luciferase gene that required a −1 frameshift within the 68-nt fragment for expression.
Giardia lamblia
trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or −1 frame, located directly before or after the heptamer, implicated the latter as the site for the −1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3′ end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a −1 ribosomal frameshift in the GLV transcript.
Enhanced Production of Macrophage Inflammatory Protein 2 (MIP-2) by In Vitro and In Vivo Infections with Encephalomyocarditis Virus and Modulation of Myocarditis with an Antibody against MIP-2
Authors: Kishimoto Chiharu , Kawamata Hiroshi , Sakai Shinya , Shinohara Hiromichi , Ochiai Hiroshi
Published: July 2002

Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein 2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine whether MIP-2 aggravates murine myocarditis. We examined (i) the MIP-2-producing activity of encephalomyocarditis (EMC) virus-infected cultured macrophages, (ii) serial plasma MIP-2 levels in EMC virus-induced mice by enzyme-linked immunosorbent assay, and (iii) the effects of antimouse MIP-2 monoclonal antibody (MAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264.7 macrophages. Five-week-old C
3
H/He mice were inoculated with EMC virus. Plasma MIP-2 levels were significantly elevated in mice on days 7 and 14 postinfection. Mice were injected subcutaneously with anti-MIP-2 MAb at 10 μg/day (group 2) or 100 μg/day (group 3) on days 0 to 5 and were observed until day 21. Uninfected control mice (group 1) were prepared. The survival rate was higher in the anti-MIP-2-treated group (group 3), but not in group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T-cell accumulation were less prominent in the anti-MIP-2 MAb-treated group, but not in group 2, compared to the level in the controls. MIP-2 is an important naturally occurring inflammatory cytokine in myocarditis, and anti-MIP-2 MAb treatment may prevent the inflammatory response.
Inducible Cyclic AMP Early Repressor Produces Reactivation of Latent Herpes Simplex Virus Type 1 in Neurons In Vitro
Authors: Colgin Mark A. , Smith Roderic L. , Wilcox Christine L.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1.
CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells
Authors: Singer Irwin I. , Scott Solomon , Kawka Douglas W. , Chin Jayne , Daugherty Bruce L. , DeMartino Julie A. , DiSalvo Jerry , Gould Sandra L. , Lineberger Janet E. , Malkowitz Lorraine , Miller Michael D. , Mitnaul Lyndon , Siciliano Salvatore J. , Staruch Mary Jo , Williams Hollis R. , Zweerink Hans J. , Springer Martin S.
Published: July 2002

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in smalltrans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.
Nef-Induced CD4 Downregulation: a Diacidic Sequence in Human Immunodeficiency Virus Type 1 Nef Does Not Function as a Protein Sorting Motif through Direct Binding to β-COP
Authors: Janvier K. , Craig H. , Le Gall S. , Benarous R. , Guatelli J. , Schwartz O. , Benichou S.
Published: July 2002

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the β subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63–73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with β-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to β-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to β-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.
Protective Cross-Reactive Cellular Immunity to Lethal A/Goose/Guangdong/1/96-Like H5N1 Influenza Virus Is Correlated with the Proportion of Pulmonary CD8+T Cells Expressing Gamma Interferon
Authors: Seo Sang Heui , Peiris Malik , Webster Robert G.
Published: July 2002

A/Goose/Guangdong/1/96-like H5N1 influenza viruses now circulating in southeastern China differ genetically from the H5N1 viruses transmitted to humans in 1997 but were their precursors. Here we show that the currently circulating H9N2 influenza viruses provide chickens with cross-reactive protective immunity against the currently circulating H5N1 influenza viruses and that this protective immunity is closely related to the percentage of pulmonary CD8+T cells expressing gamma interferon (IFN-γ). In vivo depletion of T-cell subsets showed that the cross-reactive immunity was mediated by T cells bearing CD8+and T-cell receptor (TCR) α/β and that the Vβ1 subset of TCR α/β T cells had a dominant role in protective immunity. The protective immunity induced by infection with H9N2 virus declined with time, lasting as long as 100 days after immunization. Shedding of A/Goose/Guangdong/1/96-like H5N1 virus by immunized chickens also increased with the passage of time and thus may play a role in the perpetuation and spread of these highly pathogenic H5N1 influenza viruses. Our findings indicate that pulmonary cellular immunity may be very important in protecting naïve natural hosts against lethal influenza viruses.
The Genomes of Sheeppox and Goatpox Viruses
Authors: Tulman E. R. , Afonso C. L. , Lu Z. , Zsak L. , Sur J.-H. , Sandybaev N. T. , Kerembekova U. Z. , Zaitsev V. L. , Kutish G. F. , Rock D. L.
Published: July 2002

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the
Capripoxvirus
genus of the
Poxviridae
, are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.
Minute Virus of Mice Small Nonstructural Protein NS2 Interacts and Colocalizes with the Smn Protein
Authors: Young Philip J. , Jensen Klaus T. , Burger Lisa R. , Pintel David J. , Lorson Christian L.
Published: July 2002

The small nonstructural protein NS2 of the minute virus of mice (MVM) is required for efficient viral replication, although its mode of action is unclear. Here we demonstrate that NS2 and survival motor neuron protein (Smn) interact in vitro and in vivo. NS2 and Smn also colocalize in infected nuclei at late times following MVM infection.
Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins
Authors: Binley James M. , Sanders Rogier W. , Master Aditi , Cayanan Charmagne S. , Wiley Cheryl L. , Schiffner Linnea , Travis Bruce , Kuhmann Shawn , Burton Dennis R. , Hu Shiu-Lok , Olson William C. , Moore John P.
Published: July 2002

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.
The RNA Binding Domain of the Hantaan Virus N Protein Maps to a Central, Conserved Region
Authors: Xu Xiaolin , Severson William , Villegas Noah , Schmaljohn Connie S. , Jonsson Colleen B.
Published: July 2002

The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.
Membrane Interactions of the Tick-Borne Encephalitis Virus Fusion Protein E at Low pH
Authors: Stiasny Karin , Allison Steven L. , Schalich Juliane , Heinz Franz X.
Published: July 2002

Membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic pH of the endosome and is mediated by envelope protein E, a class II viral fusion protein. The low-pH trigger induces an oligomeric rearrangement in which the subunits of the native E homodimers dissociate and the monomeric subunits then reassociate into homotrimers. Here we provide evidence that membrane binding is mediated by the intermediate monomeric form of E, generated by low-pH-induced dissociation of the dimer. Liposome coflotation experiments revealed that association with target membranes occurred only when liposomes were present at the time of acidification, whereas pretreating virions at low pH in the absence of membranes resulted in the loss of their ability to stably attach to liposomes. With the cleavable cross-linker ethylene glycolbis(succinimidylsuccinate), it was shown that a truncated soluble form of the E protein (sE) could bind to membranes only when the dimers were free to dissociate at low pH, and binding could be blocked by a monoclonal antibody that recognizes the fusion peptide, which is at the distal tip of the E monomer but is buried in the native dimer. Surprisingly, analysis of the membrane-associated sE proteins revealed that they had formed trimers. This was unexpected because this protein lacks a sequence element in the C-terminal stem-anchor region, which was shown to be essential for trimerization in the absence of a target membrane. It can therefore be concluded that the formation of a trimeric form of sE is facilitated by membrane binding. Its stability is apparently maintained by contacts between the ectodomains only and is not dependent on sequence elements in the stem-anchor region as previously assumed.
Molecular Organization of Mason-Pfizer Monkey Virus Capsids Assembled from Gag Polyprotein in
Escherichia coli
Authors: Nermut Milan V. , Bron Patrick , Thomas Daniel , Rumlova Michaela , Ruml Tomas , Hunter Eric
Published: July 2002

We describe the results of a study by electron microscopy and image processing of Gag protein shells—immature capsids—of Mason-Pfizer monkey virus assembled in
Escherichia coli
from two truncated forms of the Gag precursor: Δp4Gag, in which the C-terminal p4Gag was deleted, and Pro(−)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Δp4Gag capsids and 7.4 nm in Pro(−)CA.NC capsids. Image processing of Δp4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(−)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Δp4Gag or Pro(−)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.
Fluorescence Reagents for Labelling of Biomolecules. Part II. Reactions of 9-Isothiocyanatoacridine with Amino Acids
Authors: Podhradský Dušan , Oravec Peter , Antalík Marián , Kristian Pavol
Published: July 2002

N-(9-Acridinylthiocarbamoyl)amino acids (ATC-AA) II - VII were synthesized by reaction of amino acids with 9-isothiocyanatoacridine I, a new fluorescence labelling agent. The amino acid derivatives II - VII show high relative fluorescence, which is suitable for the determination of nanomolar amounts of ATC-AA. The kinetic measurements show that reaction of I with amino acids is 6 to 22 times faster than analogous reaction of phenyl isothiocyanate. The possibility of using 9-isothiocyanatoacridine for structure determination of proteins is discussed.
TPD Study of Carbon Monoxide Interaction with Pd/Nb Bimetallic System
Authors: Jirsak Tomas , Nikolajenko Vladimír , Knor Zlatko
Published: July 2002

Chemisorption properties of thin films of Pd evaporated onto Nb recrystallized-foil surface have been studied by temperature-programmed desorption (TPD) and by measurement of the work function changes (WFC) at room temperature. TPD results indicate two states of CO adsorbed in dependence on the thickness of the palladium layer. The low temperature state (330 K) occurs at low Pd coverages (about one monolayer). Second state, occurring at slightly higher temperature (370 K), is attributed to bulk Pd. WFC data show negligible change of the work function after deposition of small amounts of Pd onto a clean Nb surface. With increasing amount of Pd, ∆ϕ rises and reaches a saturation value. Adsorption of CO onto Pd/Nb surface caused the work function increase at all coverages of Pd deposited.