bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications
Authors: Banfield Bruce W. , Kaufman Jessica D. , Randall Jessica A. , Pickard Gary E.
Published: August 2003

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.
Magnitude and Diversity of Cytotoxic-T-Lymphocyte Responses Elicited by Multiepitope DNA Vaccination in Rhesus Monkeys
Authors: Subbramanian Ramu A. , Kuroda Marcelo J. , Charini William A. , Barouch Dan H. , Costantino Cristina , Santra Sampa , Schmitz Jörn E. , Martin Kristi L. , Lifton Michelle A. , Gorgone Darci A. , Shiver John W. , Letvin Norman L.
Published: August 2003

In an effort to develop an AIDS vaccine that elicits high-frequency cytotoxic-T-lymphocyte (CTL) responses with specificity for a diversity of viral epitopes, we explored two prototype multiepitope plasmid DNA vaccines in the simian-human immunodeficiency virus/rhesus monkey model to determine their efficiency in priming for such immune responses. While a simple multiepitope vaccine construct demonstrated limited immunogenicity in monkeys, this same multiepitope genetic sequence inserted into an immunogenic simian immunodeficiency virusgagDNA vaccine elicited high-frequency CTL responses specific for all of the epitopes included in the vaccine. Both multiepitope vaccine prototypes primed for robust epitope-specific CTL responses that developed following boosting with recombinant modified vaccinia virus Ankara vaccines expressing complete viral proteins. The natural hierarchy of immunodominance for these epitopes was clearly evident in the boosted monkeys. These studies suggest that multiepitope plasmid DNA vaccine-based prime-boost regimens can efficiently prime for CTL responses of increased breadth and magnitude, although they do not overcome predicted hierarchies of immunodominance.
Analysis of Early Human Immunodeficiency Virus Type 1 DNA Synthesis by Use of a New Sensitive Assay for Quantifying Integrated Provirus
Authors: Brussel Audrey , Sonigo Pierre
Published: August 2003

A novel
Alu
-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.
Murine Cytomegalovirus Infection Inhibits Tumor Necrosis Factor Alpha Responses in Primary Macrophages
Authors: Popkin Daniel L. , Virgin Herbert W.
Published: August 2003

Despite robust host immune responses the betaherpesvirus murine cytomegalovirus (MCMV) is able to establish lifelong infection. This capacity is due at least in part to the virus utilizing multiple immune evasion mechanisms to blunt host responses. Macrophages are an important cell for MCMV infection, dissemination, and latency despite expression in the host of multiple cytokines, including tumor necrosis factor alpha (TNF-α), that can induce an antiviral state in macrophages or other cells. In this study, we found that MCMV infection of bone marrow-derived macrophages inhibited TNF-α-induced ICAM-1 surface expression and mRNA expression in infected cells via expression of immediate early and/or early viral genes. MCMV infection blocked TNF-α-induced nuclear translocation of NF-κB. This inhibition of TNF-α signaling was explained by a decrease in TNF receptor 1 (TNFR1) and TNFR2 that was due to decreased mRNA for the latter. These findings provide a mechanism by which MCMV can evade the effects of an important host cytokine in macrophages.
Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp
111
Authors: Blanchard Emmanuelle , Hourioux Christophe , Brand Denys , Ait-Goughoulte Malika , Moreau Alain , Trassard Sylvie , Sizaret Pierre-Yves , Dubois Frederic , Roingeard Philippe
Published: August 2003

In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.
Genetic Variability of Immunomodulatory Genes in Ectromelia Virus Isolates Detected by Denaturing High-Performance Liquid Chromatography
Authors: Ribas Gloria , Rivera Jose , Saraiva Margarida , Campbell R. Duncan , Alcami Antonio
Published: August 2003

The genetic variability of nine genes in 12 isolates and strains of ectromelia virus, which causes a smallpox-like disease (mousepox) in mice, was determined and allows for classification of ectromelia viruses. The low genetic variability suggests that evolutionary pressure maintains the activity of immunomodulatory genes in natural poxvirus infections.
3′ to 5′ Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase Modulates Its Strand Displacement Activity
Authors: Zhu Yali , Trego Kelly S. , Song Liping , Parris Deborah S.
Published: August 2003

Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant strand displacement activity with or without its processivity factor, UL42. However, an exonuclease-deficient (exo
−
) pol (D368A) was capable of slow strand displacement. Although UL42 increased the rate (2/s) and processivity of strand displacement by exo
−
pol, the rate was slower than that for gap-filling synthesis. High inherent excision rates on matched primer-templates and rapid idling-turnover (successive rounds of excision and polymerization) of exo-proficient polymerases correlated with poor strand displacement activity. The results suggest that the exo activity of HSV-1 pol modulates its ability to engage in strand displacement, a function that may be important to the viability and genome stability of the virus.
Dephosphorylation of eIF-2α Mediated by the γ
1
34.5 Protein of Herpes Simplex Virus Type 1 Is Required for Viral Response to Interferon but Is Not Sufficient for Efficient Viral Replication
Authors: Cheng Guofeng , Yang Kui , He Bin
Published: August 2003

The γ
1
34.5 protein of herpes simplex virus type 1 (HSV-1) functions to block the shutoff of protein synthesis involving double-stranded RNA-dependent protein kinase (PKR). In this process, the γ
1
34.5 protein recruits cellular protein phosphatase 1 (PP1) to form a high-molecular-weight complex that dephosphorylates eIF-2α. Here we show that the γ
1
34.5 protein is capable of mediating eIF-2α dephosphorylation without any other viral proteins. While deletion of amino acids 1 to 52 from the γ
1
34.5 protein has no effect on eIF-2α dephosphorylation, further truncations up to amino acid 146 dramatically reduce the activity of the γ
1
34.5 protein. An additional truncation up to amino acid 188 is deleterious, indicating that the carboxyl-terminal domain alone is not functional. Like wild-type HSV-1, the γ
1
34.5 mutant with a truncation of amino acids 1 to 52 is resistant to interferon, and resistance to interferon is coupled to eIF-2α dephosphorylation. Intriguingly, this mutant exhibits a similar growth defect seen for the γ
1
34.5 null mutant in infected cells. Restoration of the wild-type γ
1
34.5 gene in the recombinant completely reverses the phenotype. These results indicate that eIF-2α dephosphorylation mediated by the γ
1
34.5 protein is required for HSV response to interferon but is not sufficient for viral replication. Additional functions or activities of the γ
1
34.5 protein contribute to efficient viral infection.
Identification of White Spot Syndrome Virus Latency-Related Genes in Specific-Pathogen-Free Shrimps by Use of a Microarray
Authors: Khadijah Siti , Neo Soek Ying , Hossain M. S. , Miller Lance D. , Mathavan S. , Kwang Jimmy
Published: August 2003

To investigate whether specific-pathogen-free (SPF) shrimps are asymptomatic carriers of white spot syndrome virus (WSSV), we used a WSSV-specific DNA microarray to measure WSSV gene expression in SPF and WSSV-infected shrimps. Three WSSV genes were found to be relatively highly expressed in SPF shrimps. Reverse transcription-PCR using nested primers as well as real-time detection confirmed that these genes have no detectable counterparts in GenBank; structural analysis of the putative proteins revealed helix-loop-helix and leucine zipper motifs. Viral sequences could be PCR amplified from genomic DNA of SPF shrimp, further supporting the suggestion that these shrimps are asymptomatic carriers.
Interleukin-15 and Natural Killer and NKT Cells Play a Critical Role in Innate Protection against Genital Herpes Simplex Virus Type 2 Infection
Authors: Ashkar Ali A. , Rosenthal Kenneth L.
Published: August 2003

Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15−/−(NK−and NKT−/+) mice and RAG-2−/−/γc−/−(NK−and NKT−) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15−/−and RAG-2−/−/γc−/−mice were 100-fold more susceptible and RAG-2−/−, CD-1−/−(NKT−), and gamma interferon (IFN-γ)−/−mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-γ in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2.
Parameters Driving the Selection of Nelfinavir-Resistant Human Immunodeficiency Virus Type 1 Variants
Authors: Perrin Virginie , Mammano Fabrizio
Published: August 2003

We investigated the parameters driving nelfinavir resistance, along the D30N and L90M evolutionary pathways. The advantage of the D30N mutant was mostly due to its resistance level, while the L90M mutation allowed preservation of infectivity coupled with minimal resistance. Emergence of secondary mutations further increased the selective advantage of viruses harboring D30N.
Productive Lytic Replication of a Recombinant Kaposi's Sarcoma-Associated Herpesvirus in Efficient Primary Infection of Primary Human Endothelial Cells
Authors: Gao Shou-Jiang , Deng Jian-Hong , Zhou Fu-Chun
Published: August 2003

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol.
76:
6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS.
Packaging of Brome Mosaic Virus RNA3 Is Mediated through a Bipartite Signal
Authors: Choi Yoon Gi , Rao A. L. N.
Published: August 2003

The three genomic and a single subgenomic RNA of brome mosaic virus (BMV), an RNA virus infecting plants, are packaged by a single-coat protein (CP) into three morphologically indistinguishable icosahedral virions with T = 3 quasi-symmetry. Genomic RNAs 1 and 2 are packaged individually into separate particles whereas genomic RNA3 and subgenomic RNA4 (coat protein mRNA) are copackaged into a single particle. We report here that packaging of dicistronic RNA3 requires a bipartite signal. A highly conserved 3′ tRNA-like structure postulated to function as a nucleating element (NE) for CP subunits (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA
99:
655-660, 2002) and a
cis
-acting, position-dependent packaging element (PE) of 187 nt present in the nonstructural movement protein gene are the integral components of the packaging core. Efficient incorporation into BMV virions of nonviral RNA chimeras containing NE and the PE provides confirmatory evidence that these two elements are sufficient to direct packaging. Analysis of virion RNA profiles obtained from barley protoplasts transfected with a RNA3 variant lacking the PE provides the first genetic evidence that de novo synthesized RNA4 is incompetent for autonomous assembly whereas prior packaging of RNA3 is a prerequisite for RNA4 to copackage.
Identification of a Novel Cellular Transcriptional Repressor Interacting with the Latent Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Pan Hong-Yi , Zhang Yan-Jin , Wang Xin-Ping , Deng Jian-Hong , Zhou Fu-Chun , Gao Shou-Jiang
Published: August 2003

The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione
S
-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5′-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5′-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.
Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation
Authors: Shmulevitz Maya , Salsman Jayme , Duncan Roy
Published: August 2003

Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.
Structure of Swine Vesicular Disease Virus: Mapping of Changes Occurring during Adaptation of Human Coxsackie B5 Virus To Infect Swine
Authors: Verdaguer Núria , Jimenez-Clavero Miguel A. , Fita Ignacio , Ley Victoria
Published: August 2003

The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 β-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.
Multigene RNA Vector Based on Coronavirus Transcription
Authors: Thiel Volker , Karl Nadja , Schelle Barbara , Disterer Petra , Klagge Ingo , Siddell Stuart G.
Published: August 2003

Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5′ and 3′ ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.
In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses
Authors: Tani Hideki , Limn Chang Kwang , Yap Chan Choo , Onishi Masayoshi , Nozaki Masami , Nishimune Yoshitake , Okahashi Nobuo , Kitagawa Yoshinori , Watanabe Rie , Mochizuki Rika , Moriishi Kohji , Matsuura Yoshiharu
Published: August 2003

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.
The Cap Region of Topoisomerase I Binds to Sites near Both Ends of Simian Virus 40 T Antigen
Authors: Roy Rupa , Trowbridge Pamela , Yang Zheng , Champoux James J. , Simmons Daniel T.
Published: August 2003

Two independent binding sites on simian virus 40 (SV40) T antigen for topoisomerase I (topo I) were identified. One was mapped to the N-terminal domain (residues 83 to 160) by a combination of enzyme-linked immunosorbent assays (ELISAs) and glutathione
S
-transferase (GST) pull-down assays performed with various T antigen deletion mutants. The second was mapped to the C-terminal domain (residues 602 to 708). The region in human topo I that binds to both sites in T antigen was identified by ELISAs, GST pull-down assays, and double-hexamer binding assays with topo I deletion mutants. This region corresponds to a distinct domain on topo I known as the cap region that maps from residues 175 to 433. By combining these data with information about the structure of T-antigen double hexamers associated with origin DNA, we propose that the cap region of topo I associates specifically with both ends of the double hexamer bound to the SV40 origin to initiate DNA replication.
Specific Inhibition of the Synthesis of Human Lysyl-tRNA Synthetase Results in Decreases in tRNA
Lys
Incorporation, tRNA
3
Lys
Annealing to Viral RNA, and Viral Infectivity in Human Immunodeficiency Virus Type 1
Authors: Guo Fei , Cen Shan , Niu Meijuan , Javanbakht Hassan , Kleiman Lawrence
Published: August 2003

The major human tRNA
Lys
isoacceptors,

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{1,2}^{Lys}\) \end{document}

and

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA
Lys
, via its interaction with Gag, and is a strong candidate for being the signal that specifically targets tRNA
Lys
for viral incorporation. We have transfected 293T cells with HIV-1 proviral DNA and short interfering RNA (siRNA) specific for LysRS to study the effect of diminished cellular LysRS upon tRNA
Lys
packaging,

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

annealing to viral genomic RNA, and viral production and infectivity. At early time points after siRNA transfection, an 80% inhibition of LysRS incorporation into viruses reflects an 80% reduction of newly synthesized LysRS, rather than a more limited 20 to 25% decrease in the concentration of total cell LysRS, indicating that newly synthesized LysRS in the cell may be the main source of viral LysRS. Viruses produced from cells transfected with siRNA show reduced tRNA
Lys
packaging, reduced

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}B\) \end{document}

annealing to viral RNA, and reduced viral infectivity.
Mucosal Immunization with Virus-Like Particles of Simian Immunodeficiency Virus Conjugated with Cholera Toxin Subunit B
Authors: Kang Sang-Moo , Yao Qizhi , Guo Lizheng , Compans Richard W.
Published: August 2003

To enhance the efficiency of antigen uptake at mucosal surfaces, CTB was conjugated to simian immunodeficiency virus (SIV) virus-like particles (VLPs). We characterized the immune responses to the Env and Gag proteins after intranasal administration. Intranasal immunization with a mixture of VLPs and CTB as an adjuvant elicited higher levels of SIV gp160-specific immunoglobulin G (IgG) in sera and IgA in mucosae, including saliva, vaginal-wash samples, lung, and intestine, as well as a higher level of neutralization activities than immunization with VLPs alone. Conjugation of CTB to VLPs also enhanced the SIV VLP-specific antibodies in sera and in mucosae to similar levels. Interestingly, CTB-conjugated VLPs showed higher levels of cytokine (gamma interferon)-producing splenocytes and cytotoxic-T-lymphocyte activities of immune cells than VLPs plus CTB, as well as an increased level of both IgG1 and IgG2a serum antibodies, which indicates enhancement of both Th1- and Th2-type cellular immune responses. These results demonstrate that CTB can be an effective mucosal adjuvant in the context of VLPs to induce enhanced humoral, as well as cellular, immune responses.
Enhanced Disease and Pulmonary Eosinophilia Associated with Formalin-Inactivated Respiratory Syncytial Virus Vaccination Are Linked to G Glycoprotein CX3C-CX3CR1 Interaction and Expression of Substance P
Authors: Haynes Lia M. , Jones Les P. , Barskey Albert , Anderson Larry J. , Tripp Ralph A.
Published: August 2003

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine or RSV G glycoprotein results in enhanced pulmonary disease after live RSV infection. Enhanced pulmonary disease is characterized by pulmonary eosinophilia and is associated with a substantial inflammatory response. We show that the absence of the G glycoprotein or G glycoprotein CX3C motif during FI-RSV vaccination or RSV challenge of FI-RSV-vaccinated mice, or treatment with anti-substance P or anti-CX3CR1 antibodies, reduces or eliminates enhanced pulmonary disease, modifies T-cell receptor Vβ usage, and alters CC and CXC chemokine expression. These data suggest that the G glycoprotein, and in particular the G glycoprotein CX3C motif, is key in the enhanced inflammatory response to FI-RSV vaccination, possibly through the induction of substance P.
Prolonged Exposure to Progesterone Prevents Induction of Protective Mucosal Responses following Intravaginal Immunization with Attenuated Herpes Simplex Virus Type 2
Authors: Gillgrass Amy E. , Ashkar Ali A. , Rosenthal Kenneth L. , Kaushic Charu
Published: August 2003

Depo-Provera (Depo) is a long-acting progestational formulation that is a popular form of contraception for women. In animal models of sexually transmitted diseases, it is used to facilitate infection. Here we report that treatment with Depo, in a mouse model of genital herpes simplex virus type 2 (HSV-2), altered immune responses depending on the length of time that animals were exposed to Depo prior to immunization. Mice immunized intravaginally (i.vag.) with an attenuated strain (TK
−
) of HSV-2 following longer (15 days) exposure to Depo (Depo 15 group) failed to show protection when challenged with wild-type HSV-2. In contrast, mice that were immunized shortly after Depo treatment (5 days; Depo 5 group) were fully protected and showed no genital pathology after HSV-2 challenge. High viral titers were detected in the vaginal washes of the Depo 15 group up to 6 days postchallenge. In contrast, no viral shedding was observed beyond day 3 postchallenge in the Depo 5 group. Following i.vag. TK
−
immunization, high levels of gamma interferon (IFN-γ) were detected locally in vaginal washes of the Depo 5 group but not the Depo 15 group. After HSV-2 challenge, an early peak of IFN-γ in the Depo 5 group coincided with clearance of the virus. In Depo 15 animals IFN-γ was present throughout the 6 days postinfection. HSV-2-specific T-cell cytokine responses measured in the lymph node cells of Depo 5 TK
−
-immunized mice indicated a significantly higher Th1 response than that of Depo 15 TK
−
-immunized mice. The protection after HSV-2 challenge in the Depo 5 group correlated with increased local HSV-2 glycoprotein B (gB)-specific immunoglobulin G (IgG) and IgA responses seen in the vaginal secretions. The Depo 15 group had poor gB-specific antibody responses in the genital tract after HSV-2 challenge. These results indicate that longer exposure to Depo leads to poor innate and adaptive immune responses to HSV-2 that fail to protect mice from subsequent genital challenges.
Human Papillomavirus Type 16 E6 Activates TERT Gene Transcription through Induction of c-Myc and Release of USF-Mediated Repression
Authors: McMurray H. R. , McCance D. J.
Published: August 2003

Human papillomavirus type 16 (HPV-16), a DNA tumor virus, has a causal role in cervical cancer, and the viral oncoproteins E6 and E7 contribute to oncogenesis in multiple ways. E6 increases telomerase activity in keratinocytes through increased transcription of the telomerase catalytic subunit gene (TERT), but the factors involved in this have been elusive. We have found that mutation of the proximal E box in the TERT promoter has an activating effect in luciferase assays. This suggested that a repressive complex might be present at this site. HPV-16 E6 activated the TERT promoter predominantly through the proximal E box, and thus, might be acting on this repressive complex. This site is specific for the Myc/Mad/Max transcription factors as well as USF1 and USF2. Addition of exogenous USF1 or USF2 repressed activation of the TERT promoter by E6, dependent on the proximal E box. Using siRNA against USF1 or USF2 allowed for greater activation of the TERT promoter by E6. Conversely, loss of c-Myc function, through a dominant-negative Myc molecule, reduced activation by E6. Chromatin immunoprecipitations showed that in the presence of E6, there was a reduction in binding of USF1 and USF2 at the TERT promoter proximal E box, and a concomitant increase in c-Myc bound to this site. This shows that a repressive complex containing USF1 and USF2 is present in normal cells with little or no telomerase activity. In E6 keratinocytes, this repressive complex is replaced by c-Myc, which corresponds to higher levels of TERT transcription and consequently, telomerase activity.
Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids
Authors: Newcomb William W. , Thomsen Darrell R. , Homa Fred L. , Brown Jay C.
Published: August 2003

The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 U
L
6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, U
L
26.5. Soluble U
L
26.5-portal complexes were found to assemble when purified portals were mixed in vitro with U
L
26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter “scaffold particles” formed from purified U
L
26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and U
L
26.5 combine in various proportions, with the highest observed U
L
6 content corresponding to two or three portals per scaffold particle. Association between the portal and U
L
26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including U
L
6, at a level corresponding to ∼1 portal complex per capsid. The results support the view that U
L
6 becomes incorporated into nascent HSV-1 capsids by way of a complex with U
L
26.5 and suggest further that U
L
6 may be introduced into the growing capsid as an intact portal.
Transactivation of a Viral Target Gene by Herpes Simplex Virus ICP27 Is Posttranscriptional and Does Not Require the Endogenous Promoter or Polyadenylation Site
Authors: Perkins Keith D. , Gregonis Jennifer , Borge Sarah , Rice Stephen A.
Published: August 2003

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that stimulates viral mRNA expression from many viral delayed-early and late genes during infection. One HSV-1 late gene which is highly dependent on ICP27 during infection is that encoding the glycoprotein C (gC). Here we report that the gC gene is specifically transactivated by ICP27 in transfected Vero cells. Using various gC plasmid constructs, we show that ICP27's stimulatory effects are independent of the gC gene's endogenous promoter and polyadenylation site. This suggests that ICP27-responsive elements lie in the transcribed body of the gC gene. We also show that transactivation of the gC gene by ICP27 is independent of other viral proteins, as ICP27 alone can transactivate the gC gene when its transcription is mediated by the human cytomegalovirus immediate-early gene promoter. However, when gC gene expression is driven by its endogenous promoter, the stimulatory effect of ICP27 requires additional transactivators. To explore the level at which ICP27 transactivates the gC gene, we established stably transfected Vero cell lines that have integrated copies of the gC gene under control of the cytomegalovirus immediate-early gene promoter. These gC genes are not constitutively expressed but can be efficiently induced by HSV-1 infection. Using nuclear run-on transcription assays, we show that transcriptional induction of the stably transfected genes is ICP27 independent. In contrast, accumulation of gC mRNA is very highly dependent on ICP27. Together, these results demonstrate that ICP27 posttranscriptionally activates mRNA expression from a biologically relevant viral target gene.
New Reporter Gene-Based Replication Assay Reveals Exchangeability of Replication Factors of
Porcine Circovirus
Types 1 and 2
Authors: Mankertz Annette , Mueller Bettina , Steinfeldt Tobias , Schmitt Cornelia , Finsterbusch Tim
Published: August 2003

Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the
rep
gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep′ and that the simultaneous expression of Rep and Rep′ proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The
rep
gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep′ proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a
luc
reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep′ protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the
cis
- and
trans
-acting replication factors of the two viruses are functionally exchangeable.
Active and Passive Vaccination against Hantavirus Pulmonary Syndrome with Andes Virus M Genome Segment-Based DNA Vaccine
Authors: Custer D. M. , Thompson E. , Schmaljohn C. S. , Ksiazek T. G. , Hooper J. W.
Published: August 2003

Hantavirus pulmonary syndrome (HPS) is a rapidly progressing human disease with one of the highest case fatality rates (30 to 50%) of any acute viral disease known. There are no vaccines, effective antiviral drugs, or immunologics to prevent or treat HPS. In an attempt to develop HPS medical countermeasures, we constructed an expression plasmid, pWRG/AND-M, that contains the full-length M genome segment of Andes virus (ANDV), a South American hantavirus. Transfection experiments in cell culture indicated that both the G1 and G2 glycoproteins are expressed from pWRG/AND-M. Rhesus macaques vaccinated by gene gun with pWRG/AND-M developed remarkably high levels of neutralizing antibodies that not only neutralized ANDV but also cross-neutralized other HPS-associated hantaviruses, including Sin Nombre virus. To determine if the antibodies elicited in the monkeys could confer protection, we performed a series of passive-transfer experiments using a recently described lethal HPS animal model (i.e., adult Syrian hamsters develop HPS and die within 10 to 15 days after challenge with ANDV). When injected into hamsters 1 day before challenge, sera from the vaccinated monkeys either provided sterile protection or delayed the onset of HPS and death. When injected on day 4 or 5 after challenge, the monkey sera protected 100% of the hamsters from lethal disease. These data provide a proof of concept for a gene-based HPS vaccine and also demonstrate the potential value of a postexposure immunoprophylactic to treat individuals after exposure, or potential exposure, to these highly lethal hantaviruses.
Estimation of Population Bottlenecks during Systemic Movement of
Tobacco Mosaic Virus
in Tobacco Plants
Authors: Sacristán Soledad , Malpica José M. , Fraile Aurora , García-Arenal Fernando
Published: August 2003

More often than not, analyses of virus evolution have considered that virus populations are so large that evolution can be explained by purely deterministic models. However, virus populations could have much smaller effective numbers than the huge reported census numbers, and random genetic drift could be important in virus evolution. A reason for this would be population bottlenecks during the virus life cycle. Here we report a quantitative estimate of population bottlenecks during the systemic colonization of tobacco leaves by
Tobacco mosaic virus
(TMV). Our analysis is based on the experimental estimation of the frequency of different genotypes of TMV in the inoculated leaf, and in systemically infected leaves, of tobacco plants coinoculated with two TMV genotypes. A simple model, based on the probability that a leaf in coinoculated plants is infected by just one genotype and on the frequency of each genotype in the source, was used to estimate the effective number of founders for the populations in each leaf. Results from the analysis of three leaves per plant in plants inoculated with different combinations of three TMV genotypes yielded highly consistent estimates. Founder numbers for each leaf were small, in the order of units. This would result in effective population numbers much smaller than the census numbers and indicates that random effects due to genetic drift should be considered for understanding virus evolution within an infected plant.
Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication
Authors: Apolloni Ann , Hooker C. William , Mak Johnson , Harrich David
Published: August 2003

The human immunodeficiency virus type 1 (HIV-1) Tat protein enhances reverse transcription, but it is not known whether Tat acts directly on the reverse transcription complex or through indirect mechanisms. Since processing of Tat by HIV protease (PR) might mask its presence and, at least in part, explain this lack of data, we asked whether Tat can be cleaved by PR. We used a rabbit reticulocyte lysate (RRL) system to make Tat and PR. HIV-1 PR is expressed as a Gag-Pol fusion protein, and a PR-inactivated Gag-Pol is also expressed as a control. We showed that Tat is specifically cleaved in the presence of PR, producing a protein of approximately 5 kDa. This result suggested that the cleavage site was located in or near the Tat basic domain (amino acids 49 to 57), which we have previously shown to be important in reverse transcription. We created a panel of alanine-scanning mutations from amino acids 45 to 54 in Tat and evaluated functional parameters, including transactivation, reverse transcription, and cleavage by HIV-1 PR. We showed that amino acids 49 to 52 (RKKR) are absolutely required for Tat function in reverse transcription, that mutation of this domain blocks cleavage by HIV-1 PR, and that other pairwise mutations in this region modulate reverse transcription and proteolysis in strikingly similar degrees. Mutation of Tat Y47G48 to AA also down-regulated Tat-stimulated reverse transcription but had little effect on transactivation or proteolysis by HIV PR, suggesting that Y47 is critical for reverse transcription. We altered the
tat
gene of the laboratory strain NL4-3 to Y47D and Y47N so that overlapping reading frames were not affected and showed that Y47D greatly diminished virus replication and conveyed a reverse transcription defect. We hypothesize that a novel, cleaved form of Tat is present in the virion and that it requires Y47 for its role in support of efficient reverse transcription.
Human T-Cell Leukemia Virus Type 1 Envelope Glycoprotein gp46 Interacts with Cell Surface Heparan Sulfate Proteoglycans
Authors: Piñon Josefina D. , Klasse P. J. , Jassal Sushma R. , Welson Sandy , Weber Jonathan , Brighty David W. , Sattentau Quentin J.
Published: August 2003

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.