bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity
Authors: Miles Lesa R. , Agresta Beth E. , Khan Mahfuz B. , Tang Shixing , Levin Judith G. , Powell Michael D. 
Published: May 2005

ABSTRACT

We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSΔ
nef
. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5′ end appeared to have less of an effect on infectivity than mutation of the 3′ end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3′ end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.
Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response
Authors: Isler Jennifer A. , Skalet Alison H. , Alwine James C. 
Published: May 2005

ABSTRACT
Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.
The bZIP Transcription Factor ATFx Binds Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax and Represses HTLV-1 Long Terminal Repeat-Mediated Transcription
Authors: Forgacs Eva , Gupta Saurabh K. , Kerry Julie A. , Semmes O. John 
Published: May 2005

ABSTRACT
The human T-cell leukemia virus type 1 (HTLV-1) viral protein Tax is a transactivator of transcription driven by the cognate viral long terminal repeat (LTR). Tax exerts its effect through three nonidentical copies of the Tax-responsive element (TxRE), a member of the asymmetric cyclic AMP response element (CRE) family of enhancer sequences. Transactivation is mediated via interaction of Tax with members of the CREB/ATF family bound to TxRE. We have identified a cellular repressor of transcription, activating transcription factor x (ATFx), as a novel Tax-binding protein. In addition to binding directly to Tax we show by electrophoretic mobility shift assay that ATFx binds to the TxRE enhancer element via the bZIP domain. The functional impact of this bridging interaction results in repression of both basal and Tax-induced transcription from the HTLV-1 LTR. ATFx is unique among ATF family of proteins in that it is cell cycle regulated and exerts a tight repressive control over apoptotic signaling. We propose that recruitment of ATFx to the HTLV-1 LTR serves to link viral transcription with critical events in cellular homeostasis.
Herpes Simplex Virus Type 1 UL51 Protein Is Involved in Maturation and Egress of Virus Particles
Authors: Nozawa Naoki , Kawaguchi Yasushi , Tanaka Michiko , Kato Akihisa , Kato Ai , Kimura Hiroshi , Nishiyama Yukihiro 
Published: May 2005

ABSTRACT

The
UL51
gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the
UL51
gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.
RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles
Authors: Peters Katrin , Wiktorowicz Tatiana , Heinkelein Martin , Rethwilm Axel 
Published: May 2005

ABSTRACT

Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol.
76:
10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified
cis
-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5′ long terminal repeat and one at the 3′ end of the
pol
gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.
Diversifying Selection in Human Papillomavirus Type 16 Lineages Based on Complete Genome Analyses
Authors: Chen Zigui , Terai Masanori , Fu Leiping , Herrero Rolando , DeSalle Rob , Burk Robert D. 
Published: May 2005

ABSTRACT
Human
papillomavirus type 16 (HPV16) is the primary etiological
agent of cervical cancer, the second most common cancer in women
worldwide. Complete genomes of 12 isolates representing the major
lineages of HPV16 were cloned and sequenced from cervicovaginal cells.
The sequence variations within the open reading frames (ORFs) and
noncoding regions were identified and compared with the HPV16R
reference sequence (50).
This whole-genome approach gives us unprecedented precision in
detailing sequence-level changes that are under selection on a
whole-viral-genome scale. Of 7,908 base pair nucleotide positions, 313
(4.0%) were variable. Within the 2,452 amino acids
(aa) comprising 8 ORFs, 243 (9.9%) amino acid positions were
variable. In order to investigate the molecular evolution of HPV16
variants, maximum likelihood models of codon substitution were
used to identify lineages and amino acid sites under selective
pressure. Five codon sites in the E5 (aa 48, 65) and E6 (aa
10, 14, 83) ORFs were demonstrated to be under diversifying selective
pressure. The E5 ORF had the overall highest nonsynonymous/synonymous
substitution rate (ω) ratio (M3 = 0.7965). The E2 gene
had the next-highest ω ratio (M3 = 0.5611); however, no
specific codons were under positive selection. These data
indicate that the E6 and E5 ORFs are evolving under positive Darwinian
selection and have done so in a relatively short time period. Whether
response to selective pressure upon the E5 and E6 ORFs
contributes to the biological success of HPV16, its specific biological
niche, and/or its oncogenic potential remains to be
established.
Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding
Authors: Brown Erica L. , Lyles Douglas S. 
Published: May 2005

ABSTRACT
Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.
Cinanserin Is an Inhibitor of the 3C-Like Proteinase of Severe Acute Respiratory Syndrome Coronavirus and Strongly Reduces Virus Replication In Vitro
Authors: Chen Lili , Gui Chunshan , Luo Xiaomin , Yang Qingang , Günther Stephan , Scandella Elke , Drosten Christian , Bai Donglu , He Xichang , Ludewig Burkhard , Chen Jing , Luo Haibin , Yang Yiming , Yang Yifu , Zou Jianping , Thiel Volker , Chen Kaixian , Shen Jianhua , Shen Xu , Jiang Hualiang 
Published: May 2005

ABSTRACTThe 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLproof SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 μM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50values ranging from 19 to 34 μM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.
Characterization of Human Immunodeficiency Virus Gag-Specific Gamma Interferon-Expressing Cells following Protective Mucosal Immunization with Alphavirus Replicon Particles
Authors: Gupta Soumi , Janani Ramesh , Bin Qian , Luciw Paul , Greer Catherine , Perri Silvia , Legg Harold , Donnelly John , Barnett Susan , O'Hagan Derek , Polo John M. , Vajdy Michael 
Published: May 2005

ABSTRACTA safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed α4β7 or αEβ7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed α4β7 or CD62L than expressed αEβ7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.
Elimination of
ie
1 Significantly Attenuates Murine Cytomegalovirus Virulence but Does Not Alter Replicative Capacity in Cell Culture
Authors: Ghazal Peter , Visser Astrid E. , Gustems Montse , García Rosalía , Borst Eva Maria , Sullivan Kevin , Messerle Martin , Angulo Ana 
Published: May 2005

ABSTRACT

The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the
ie
1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the
ie
1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 × 10
4
PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of
ie
1. These results demonstrate the significance of the
ie
1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the
ie
1 gene.
Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity
Authors: Mérai Zsuzsanna , Kerényi Zoltán , Molnár Attila , Barta Endre , Válóczi Anna , Bisztray György , Havelda Zoltán , Burgyán József , Silhavy Dániel 
Published: May 2005

ABSTRACT

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus
Tombusvirus
. We show that the
Pothos latent virus
(PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.
Loss of Interferon Regulatory Factor 3 in Cells Infected with Classical Swine Fever Virus Involves the N-Terminal Protease, N
pro
Authors: La Rocca S. Anna , Herbert Rebecca J. , Crooke Helen , Drew Trevor W. , Wileman Thomas E. , Powell Penny P. 
Published: May 2005

ABSTRACT

We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (N
pro
) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from N
pro
-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.
Heat Shock Protein 90 Expression in Epstein-Barr Virus-Infected B Cells Promotes γδ T-Cell Proliferation In Vitro
Authors: Kotsiopriftis Maria , Tanner Jerome E. , Alfieri Caroline 
Published: May 2005

ABSTRACTThe aim of this study was to elucidate the in vitro response of γδ T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as γδ T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that γδ T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of αβ T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited γδ T-cell expansion by 92%. The inhibition of γδ T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of γδ T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of γδ T cells during acute EBV infection.