Life Sciences Commons^™

Rdr6 Has A Broad-Spectrum But Temperature-Dependent Antiviral Defense Role In Nicotiana Benthamiana, Feng Qu, Xiaohong Ye, Guichuan Hou, Shirley Sato, Thomas E. Clemente, Thomas Jack Morris

Nebraska Center for Virology: Faculty Publications

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested …

Aids And Associated Malignancies, Charles Wood, William Harrington Jr.

Nebraska Center for Virology: Faculty Publications

AIDS associated malignancies (ARL) is a major complication associated with AIDS patients upon immunosuppression. Chronically immunocompromised patients have a markedly increased risk of developing lymphoproliferative disease. In the era of potent antiretrovirals therapy (ARV), the malignant complications due to HIV-I infection have decreased in developed nations where ARV is administered, but still poses a major problem in developing countries where HIV-l incidence is high and ARV is still not yet widely available. Even in ARV treated individuals there is a concern that the prolonged survival of many HIV-l carriers is likely to eventually result in an increased number of malignancies …

Interspecies Transmission Of Chronic Wasting Disease Prions To Squirrel Monkeys (Saimiri Sciureus), Richard F. Marsh, Anthony E. Kincaid, Richard A. Bessen, Jason C. Bartz

Nebraska Center for Virology: Faculty Publications

Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.

Suppressors Of Rna Silencing Encoded By Plant Viruses And Their Role In Viral Infections, Feng Qu, Thomas Jack Morris

Nebraska Center for Virology: Faculty Publications

Published in FEBS Letters 579:26 (October 31, 2005), pp. 5958–5964; doi 10.1016/j.febslet.2005.08.041 Copyright © 2005 Federation of European Biochemical Societies; published by Elsevier B.V. Used by permission. http://www.sciencedirect.com/science/journal/00145793

Extraneural Prion Neuroinvasion Without Lymphoreticular System Infection, Jason C. Bartz, Crista Dejoia, Tammy Tucker, Anthony E. Kincaid, Richard A. Bessen

Nebraska Center for Virology: Faculty Publications

While prion infection of the lymphoreticular system (LRS) is necessary for neuroinvasion in many prion diseases, in bovine spongiform encephalopathy and atypical cases of sheep scrapie there is evidence to challenge that LRS infection is required for neuroinvasion. Here we investigated the role of prion infection of LRS tissues in neuroinvasion following extraneural inoculation with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. DY TME agent infectivity was not detected in spleen or lymph nodes following intraperitoneal inoculation and clinical disease was not observed following inoculation into the peritoneum or lymph nodes, or after oral ingestion. …

Functional Mimicry Of A Human Immunodeficiency Virus Type 1 Coreceptor By A Neutralizing Monoclonal Antibody, Shi-Hua Xiang, Michael Farzan, Zhihai Si, Navid Madani, Liping Wang, Eric Rosenberg, James Robinson, Joseph Sodroski

Nebraska Center for Virology: Faculty Publications

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 β19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 …

Kaposi’S Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Replication And Transcription Activator Regulates Viral And Cellular Genes Via Interferon-Stimulated Response Elements, Jun Zhang, Jinzhang Wang, Charles Wood, Dongsheng Xu, Luwen Zhang

Nebraska Center for Virology: Faculty Publications

Kaposi’s sarcoma-associated herpesvirus (also called human herpesvirus 8 [HHV-8]) replication and transcription activator (RTA) is apparently necessary and sufficient for the switch from viral latency to lytic replication. RTA may regulate open reading frame (ORF) K14 (viral OX-2 homologue) and ORF74 (viral G-protein-coupled receptor homologue) genes through an interferon-stimulated response element (ISRE)-like sequence (K14 ISRE) in the promoter region. RTA strongly activated a K14 ISRE-containing K14-ORF74 promoter reporter construct and a heterologous promoter reporter construct containing K14 ISRE. RTA could bind to K14 ISRE and other ISREs, activate promoter reporter constructs from interferon-simulated genes (ISGs), and selectively induce three endogenous …

Identification Of Cis-Acting Elements That Mediate The Replication And Maintenance Of Human Papillomavirus Type 16 Genomes In Saccharomyces Cerevisiae, Kitai Kim, Peter C. Angeletti, Elizabeth C. Hassebroek, Paul F. Lambert

Nebraska Center for Virology: Faculty Publications

Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote, Saccharomyces cerevisiae. Specifically, papillomavirus …

Comparative Genomics Of Foot-And-Mouth Disease Virus, C. Carrillo, E. R. Tuhlman, Gustavo A. Delhon, Z. Lu, A. Carreno, A. Vagnozzi, G. F. Kutish, D. L. Rock

Nebraska Center for Virology: Faculty Publications

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5’ and 3’ untranslated regions (UTR), as was tolerance for insertions/deletions in the 5’ UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for …

Modulation Of Human Herpesvirus 8/Kaposi’S Sarcoma-Associated Herpesvirus Replication And Transcription Activator Transactivation By Interferon Regulatory Factor 7, Jinzhang Wang, Jun Zhang, William Harrington Jr., John T. West, Charles Wood

Nebraska Center for Virology: Faculty Publications

Human herpesvirus 8 (HHV-8)/Kaposi’s sarcoma-associated herpesvirus infection goes through lytic and latent phases that are regulated by viral gene products, but very little is known about the involvement of host proteins. The replication and transcription activator (RTA) is a viral protein sufficient to initiate lytic replication by activating downstream genes, including the viral early gene open reading frame 57 (ORF 57), which codes for a posttranscriptional activator. In this study, we demonstrate that cellular interferon regulatory factor 7 (IRF-7) negatively regulates this process by competing with RTA for binding to the RTA response element in the ORF 57 promoter to …

Human Dendritic Cells Transduced With Herpes Simplex Virus Amplicons Encoding Human Immunodeficiency Virus Type 1 (Hiv-1) Gp120 Elicit Adaptive Immune Responses From Human Cells Engrafted Into Nod/Scid Mice And Confer Partial Protection Against Hiv-1 Challenge, Santhi Gorantla, Kathlyn Santos, Vakara Meyer, Stephen Dewhurst, William J. Bowers, Howard J. Federoff, Howard Gendelman, Larisa Poluektova

Nebraska Center for Virology: Faculty Publications

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replicationdefective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent …

In Vitro And In Vivo Translational Efficiencies Of The 5′ Untranslated Region From Eight Genotype 2 Bovine Viral Diarrhea Virus Field Isolates, Christina L. Topliff, Seung K. Chon, Ruben O. Donis, Kent M. Eskridge, Clayton L. Kelling

Nebraska Center for Virology: Faculty Publications

We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of …

The Nuclear Localization Of The Arabidopsis Transcription Factor Tip Is Blocked By Its Interaction With The Coat Protein Of Turnip Crinkle Virus, Tao Ren, Feng Qu, Thomas Jack Morris

Nebraska Center for Virology: Faculty Publications

We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift …

Replication And Encapsidation Of Papillomaviruses In Saccharomyces Cerevisiae, Peter C. Angeletti

Nebraska Center for Virology: Faculty Publications

Improvements in methodologies to recapitulate and study particular biological functions of the

papillomavirus life cycle have led to great advances in our knowledge of these viruses. Described in

this chapter are techniques that allow low-copy and high-copy replication of full-length human

papillomavirus (HPV) genomes, as well as assembly of virus-like particles, in Saccharomyces

cerevisiae (yeast). This system has several distinct advantages that make it an attractive complement

to the well-established raft-culturing system. First, yeast are inexpensive, rapid, and simple to culture

in the lab. Second, they provide an ever-widening array of genetic tools to analyze HPV functions

—most recently notable, …

Antigenicity And Immunogenicity Of A Synthetic Human Immunodeficiency Virus Type 1 Group M Consensus Envelope Glycoprotein, Feng Gao, Eric A. Weaver, Zhongjing Lu, Yingying Li, Hua-Xin Liao, Benjiang Ma, S. Munir Alam, Richard M. Scearce, Laura L. Sutherland, Jae-Sung Yu, Julie M. Decker, George M. Shaw, David C. Montefiori, Bette T. Korber, Beatrice H. Hahn, Barton F. Haynes

Nebraska Center for Virology: Faculty Publications

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 …

Antigenicity And Immunogenicity Of A Synthetic Human Immunodeficiency Virus Type 1 Group M Consensus Envelope Glycoprotein, Feng Gao, Eric A. Weaver, Zhongjing Lu, Yingying Li, Hua-Xin Liao, Benjiang Ma, S. Munir Alam, Richard M. Scearce, Laura L. Sutherland, Jae-Sung Yu, Julie M. Decker, George M. Shaw, David C. Montefiori, Bette T. Korber, Beatrice H. Hahn, Barton F. Haynes

Nebraska Center for Virology: Faculty Publications

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 …

Genome Of Deerpox Virus, C. L. Afonso, Gustavo A. Delhon, E. R. Tuhlman, Z. Lu, A. Zsak, V. M. Becerra, L. Zsak, G. F. Kutish, D. L. Rock

Nebraska Center for Virology: Faculty Publications

Deerpox virus (DPV), an uncharacterized and unclassified member of the Poxviridae, has been isolated from North American free-ranging mule deer (Odocoileus hemionus) exhibiting mucocutaneous disease. Here we report the genomic sequence and comparative analysis of two pathogenic DPV isolates, W-848-83 (W83) and W-1170-84 (W84). The W83 and W84 genomes are 166 and 170 kbp, containing 169 and 170 putative genes, respectively. Nucleotide identity between DPVs is 95% over the central 157 kbp. W83 and W84 share similar gene orders and code for similar replicative, structural, virulence, and host range functions. DPV open reading frames (ORFs) with putative …

Evolution Of Subtype C Hiv-1 Env In A Slowly Progressing Zambian Infant, Hong Zhang, Federico Hoffmann, Jun He, Xiang He, Chipepo Kankasa, Ruth Ruprecht, John T. West, Guillermo Orti, Charles Wood

Nebraska Center for Virology: Faculty Publications

Background: Given the high prevalence of mother to child infection, the development of a better understanding of African subtype C HIV-1 transmission and natural evolution is of significant importance. In this study, we genotypically and phenotypically characterized subtype C viruses isolated over a 67-month follow-up period from an in utero-infected Zambian infant. Changes in genotype and phenotype were correlated to alterations of the host humoral immune response.

Results: A comparison of baseline maternal and infant samples indicated that the infant sequences are monophyletic and contain a fraction of the diversity observed in the mother. This finding suggests that selective …

Epidemiological Characteristics Of Human Herpesvirus-8 Infection In A Large Population Of Antenatal Women In Zambia, W. Klaskala, B. P. Brayfield, C. Kankasa, G. Bhat, J. T. West, C. D. Mitchell, Charles Wood

Nebraska Center for Virology: Faculty Publications

Comprehensive data describing epidemiological characteristics of the human herpesvirus-8 or Kaposi’s sarcoma-associated herpesvirus (HHV-8 or KSHV) infection among pregnant women in a central sub-Saharan Africa are not available. This study determined virus prevalence estimates and the risk factors associated with HHV-8 infection. Cross-sectional, enrollment visit data were analyzed from a prospective cohort study of perinatal transmission of HHV-8 in Lusaka, Zambia. Exposure data were obtained via structured interview, physical examination, medical chart review, and laboratory testing. Among 3,160 antenatal women serologically screened for HHV-8 between September 1998 and October 2000, 40.2% were seropositive. The HHV-8 positive women were more likely …

Locatization Of Human Herpesvirus 8 In Human Sperms By In Situ Pcr, Omar Bagasra, Deepa Patel, Lisa Bobroski, Jamil A. Abbasi, Alex U. Bagasra, Hasna Baidouri, Twaina Harris, Albert El-Roeiy, Zsolt Lengvarszky, Homayoon Farzadegan, Charles Wood

Nebraska Center for Virology: Faculty Publications

Objectives: Defining the mechanism of infection with human herpesvirus-8 (HHV-8) or Kaposi’s sarcoma associated herpesvirus (KSHV) is an important clinical issue. HHV-8 has been linked to Kaposi’s sarcoma (KS) development in HIV-1-infected individuals, and KS develops in 40% of those infected with both viruses. A series of epidemiological data suggest that sexual transmission is one of the routes of transmission for HHV-8. In our studies, we sought to assess the cellular reservoirs of HHV-8 DNA in the semen of HIV-1-infected men and the potential role of HHV-8 infected spermatozoa in horizontal transmission. Design and methods: A nested polymerase chain reaction …

Compartmentalization Of Hiv-1 In The Central Nervous System: Role Of The Choroid Plexus, Evan J. Burkala, Jun He, John T. West, Charles Wood, Carol K. Petito

Nebraska Center for Virology: Faculty Publications

Objectives: To determine the genetic and phenotypic composition of HIV-1 found in the choroid plexus (CPx) and its relationship to virus in the brain and peripheral lymphoid tissue. Design: Phenotypic and molecular comparisons of HIV-1 found in autopsy brain, CPx, and spleen tissues. Methods: HIV-1 was co-cultured from matched postmortem brain (basal ganglia), CPx, and spleen tissues of AIDS patients with and without HIV-associated encephalitis and dementia. Viral phenotypes were determined by infection of monocyte-derived macrophages, MT-2 or co-receptor-specific cell lines. Viral env and pol sequences were determined from genomic DNA isolated directly from tissues or co-cultures, and phylogenetic comparisons …

Guidelines For The Management Of Hiv Infection In Pregnant Women And The Prevention Of Mother-To-Child Transmission Of Hiv, David Hawkins, M. Blott, P. Clayden, A, De Ruiter, G. Foster, C. Gilling-Smith, B. Gosrani, H. Lyall, D. Mercey, M.-L. Newell, S. O'Shea, R. Smith, J. Sunderland, Charles Wood, G. Taylor

Nebraska Center for Virology: Faculty Publications

The prevalence of HIV infection amongst women giving birth in England and Wales has increased every year since 1990. Results from the Unlinked Anonymous Surveys of infection in pregnancy, show that in 2003, the prevalence reached one in 180 (0.56%) in inner London, one in 271 in outer London (0.37%) and one in 1,282 (0.08%) in the rest of England [1]. The majority of these women are from sub-Saharan Africa. The Department of Health policy of recommending an HIV test to every pregnant woman [2] has resulted in an increase in the proportion of these women who are aware of …

Dichotomy In Cross-Clade Reactivity And Neutralization By Hiv-1 Sera: Implications For Active And Passive Immunotherapy, Lisa A. Cavacini, Mark Duval, Ajay Patil, Charles Wood, Kanneth H. Mayer, Ruth M. Ruprecht, Marshall L. Posner

Nebraska Center for Virology: Faculty Publications

The identification of broadly reactive and cross-clade neutralizing antibodies will facilitate the development of a more universally effective vaccine for human immunodeficiency virus (HIV). Antibodies in sera from individuals infected with Clade B HIV bind native primary viral isolates, and virus binding correlates with neutralization and stable clinical disease. In this study, we quantified cross-clade antibody reactivity and neutralization by Clades B and C sera. Primary viral isolates were captured by serum IgG bound to anti-human IgG and quantitated as p24 released by lysis of captured virus. Neutralization was determined using PHA-stimulated PBMC. Clade B antibodies reacted more frequently with …

Proteomic Fingerprints Distinguish Microglia, Bone Marrow, And Spleen Macrophage Populations, Yoshimi Enose, Christopher J. Destache, Andrea L. Mack, James R. Anderson, Fred Ullrich, Pawel Ciborowski, Howard E. Gendelman

Nebraska Center for Virology: Faculty Publications

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological …