bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stability and Function of JC Virus Large T Antigen and T′ Proteins Are Altered by Mutation of Their Phosphorylated Threonine 125 Residues
Authors: Tyagarajan Shiva K. , Frisque Richard J. 
Published: February 2006

ABSTRACT

JC virus (JCV), a human polyomavirus, exhibits oncogenic activity in rodents and primates. The large tumor antigens (TAgs) of the polyomaviruses play key roles in viral replication and oncogenic transformation. Analyses of JCV TAg phosphorylation mutants indicated that the amino-terminal phosphorylation site at threonine 125 (T125) is critical to TAg replication function. This site is also conserved in the TAg splice variants T′
135
, T′
136
, and T′
165
. By constructing stable cell lines expressing JCV T125A and T125D mutants, we show that mutation of this phosphorylation site to alanine generates an unstable TAg; however, the stability of the three T′ proteins is unaffected. JCV T125A mutant proteins bind the retinoblastoma protein (RB) family members p107 and p130 with slightly reduced efficiencies and fail to induce the release of transcriptionally active E2F from RB-E2F complexes. On the other hand, cell lines expressing JCV T125D mutant proteins produce stable TAg and T′ proteins which bind p107 and p130 more efficiently than do the wild-type proteins. In addition, T125D mutant proteins efficiently induce the release of E2F from RB-E2F complexes. T125D mutant cell lines, unlike the T125A mutant lines, continue to grow under conditions of low serum concentration and anchorage independence. Finally, both T125A and T125D mutant viruses are replication defective. Phosphorylation of the T125 site is likely mediated by a cyclin-cyclin-dependent kinase, suggesting that JCV TAg and T′ protein functions that mediate viral replication and oncogenic transformation events are regulated in a cell cycle-dependent manner.
Human Immunodeficiency Virus (HIV) Vaccine Trials: a Novel Assay for Differential Diagnosis of HIV Infections in the Face of Vaccine-Generated Antibodies
Authors: Khurana Surender , Needham James , Mathieson Bonnie , Rodriguez-Chavez Isaac R. , Catanzaro Andrew T. , Bailer Robert T. , Kim Jerome , Polonis Vicky , Cooper David A. , Guerin Jan , Peterson Michael L. , Gurwith Marc , Nguyen Nga , Graham Barney S. , Golding Hana 
Published: February 2006

ABSTRACT
All current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and elicit antibodies that react positively in licensed HIV diagnostic tests, which contain similar viral products. Thus, vaccine trial participants could be falsely diagnosed as infected with HIV. Additionally, uninfected, seropositive vaccinees may encounter long-term social and economic harms. Moreover, this also interferes with early detection of true HIV infections during preventive HIV vaccine trials. An HIV-seropositive test result among uninfected vaccine trial participants is a major public health concern for volunteers who want to participate in future HIV vaccine trials. Based on the increased number of HIV vaccines being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Using a whole-HIV-genome phage display library, we identified conserved sequences in Env-gp41 and Gag-p6 which are recognized soon after infection, do not contain protective epitopes, and are not part of most current HIV vaccines. We established a new HIV serodetection assay based on these peptides. To date, this assay, termed HIV-SELECTEST, demonstrates >99% specificity and sensitivity. Importantly, in testing of plasma samples from multiple HIV vaccine trials, uninfected trial participants scored negative, while all intercurrent infections were detected within 1 to 3 months of HIV infection. The new HIV-SELECTEST is a simple but robust diagnostic tool for easy implementation in HIV vaccine trials and blood banks worldwide.
Retroviral Restriction Factors Fv1 and TRIM5α Act Independently and Can Compete for Incoming Virus before Reverse Transcription
Authors: Passerini Luca D. , Keckesova Zuzana , Towers Greg J. 
Published: February 2006

ABSTRACT

The restriction factors Fv1 and TRIM5α provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5α-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5α for virus before reverse transcription. In human cells expressing Fv1
b
, N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5α by Fv1. Our findings suggest that, like TRIM5α, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5α when expressed in human cells.
Mouse-Adapted Scrapie Infection of SN56 Cells: Greater Efficiency with Microsome-Associated versus Purified PrP-res
Authors: Baron Gerald S. , Magalhães Ana C. , Prado Marco A. M. , Caughey Byron 
Published: February 2006

ABSTRACTThe process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.
Hepatitis B Virus Reverse Transcriptase and ε RNA Sequences Required for Specific Interaction In Vitro
Authors: Hu Jianming , Boyer Morgan 
Published: February 2006

ABSTRACT
Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific ε binding, we have defined the protein and RNA sequences required for specific HBV RT-ε interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for ε binding. With respect to the ε RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.
Comparison of the Replication of Influenza A Viruses in Chinese Ring-Necked Pheasants and Chukar Partridges
Authors: Humberd Jennifer , Guan Yi , Webster Robert G. 
Published: February 2006

ABSTRACT
We investigated the replication and transmission of avian influenza A viruses in two species thought to be intermediate hosts in the spread of influenza A viruses in live poultry markets: Chinese ring-necked pheasants and chukar partridges. All 15 hemagglutinin subtypes replicated in pheasants, and most subtypes transmitted to naïve contact pheasants, primarily via the fecal-oral route. Many viruses were shed from the gastrointestinal tract of experimentally inoculated pheasants for 14 days or longer. Virus was isolated from the cloacal swabs of one contact pheasant for an unprecedented 45 days. Chukar partridges were less susceptible to infection with avian influenza viruses. The viruses that replicated in chukar partridges were isolated for 7 days after experimental inoculation, predominantly from the respiratory tract. We detected high neutralizing antibody titers with correspondingly low levels of serum hemagglutination inhibition antibody titers in pheasants and chukar partridges when chicken red blood cells were used in serological analyses. When horse erythrocytes were used, antibody titers were comparable to those obtained by using the neutralization assay. More importantly, the results suggested that pheasants can serve as a reservoir of influenza virus. Because of their continuous asymptomatic infection and longer stay in the markets, pheasants are ideal “carriers” of influenza A viruses. Their continued presence in live markets contributes to the perpetuation and genetic interaction of influenza viruses there. On the basis of our findings, it does not make good sense to ban quail but not pheasants from the live markets.
Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication
Authors: Serva Saulius , Nagy Peter D. 
Published: February 2006

ABSTRACT

Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (
ssa1 ssa2
) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned
ssa1 ssa2
mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.
RNA Secondary Structure in the Coding Region of Dengue Virus Type 2 Directs Translation Start Codon Selection and Is Required for Viral Replication
Authors: Clyde Karen , Harris Eva 
Published: February 2006

ABSTRACT

Dengue virus
is a positive-strand RNA virus and a member of the genus
Flavivirus
, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region
cis
element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.
Bovine Coronavirus 5′-Proximal Genomic Acceptor Hotspot for Discontinuous Transcription Is 65 Nucleotides Wide
Authors: Wu Hung-Yi , Ozdarendeli Aykut , Brian David A. 
Published: February 2006

ABSTRACT
Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5′-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of bovine coronavirus) near the 3′ end of the 5′-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable bovine coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (∼2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5′-proximal “hotspot.” Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5′-proximal multifunctional structure.
Gradual Elimination of Retroviruses in YBR/Ei Mice
Authors: MacDearmid Cameron C. , Case Laure K. , Starling Christa L. , Golovkina Tatyana V. 
Published: February 2006

ABSTRACTMouse mammary tumor virus (MMTV), a well-characterized retrovirus that causes mammary tumors in susceptible mice, is commonly used to investigate virus-host interactions. We have shown that YBR/Ei mice demonstrate a novel, dominant mechanism of resistance to MMTV infection and MMTV-induced mammary tumors. MMTV can both establish infection in YBR/Ei mice and be transmitted by YBR/Ei mice as an infectious virus. However, virus production is severely attenuated, resulting in gradual clearance of infection in successive generations. Our transfer experiments showed that T cells generated in MMTV-infected resistant mice were required to restrict MMTV replication in susceptible mice. These results emphasize the importance of inducing T-cell responses for effective protection against retroviral infections.
Heptad Repeat 2 in Herpes Simplex Virus 1 gH Interacts with Heptad Repeat 1 and Is Critical for Virus Entry and Fusion
Authors: Gianni Tatiana , Piccoli Angela , Bertucci Carlo , Campadelli-Fiume Gabriella 
Published: February 2006

ABSTRACT
Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the α-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.
Recognition of Vaccinia Virus-Infected Cells by Human Natural Killer Cells Depends on Natural Cytotoxicity Receptors
Authors: Chisholm Susan E. , Reyburn Hugh T. 
Published: February 2006

ABSTRACTNatural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.
Transcriptional Origin of Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs
Authors: Cai Xuezhong , Cullen Bryan R. 
Published: February 2006

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.
Latency-Associated Nuclear Antigen (LANA) of Kaposi's Sarcoma-Associated Herpesvirus Interacts with Origin Recognition Complexes at the LANA Binding Sequence within the Terminal Repeats
Authors: Verma Subhash C. , Choudhuri Tathagata , Kaul Rajeev , Robertson Erle S. 
Published: February 2006

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the
cis-
acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G
1
/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.