Copyright (c) 2009 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/virologypub Recent documents in Virology Papers en-us Fri, 30 Oct 2009 23:21:55 PDT 3600 http://digitalcommons.unl.edu/virologypub/197 http://digitalcommons.unl.edu/virologypub/197 Thu, 29 Oct 2009 12:22:47 PDT K-RBP is a KRAB-containing zinc finger protein with multiple zinc finger motifs and represses Kaposi's sarcoma-associated herpesvirus (KSHV) transactivator RTA-mediated transactivation of several viral lytic gene promoters, including the ORF57 promoter. Whether K-RBP binds DNA through its zinc fingers and the role of zinc finger domain in repressing gene expression are unclear. Here we report that K-RBP binds DNA through its zinc finger domain and the target DNA sequences contain high GC content. Furthermore, K-RBP binds to KSHV ORF57 promoter, which contains a GC-rich motif. K-RBP suppresses the basal ORF57 promoter activity as well as RTA-mediated activation. The zinc finger domain of K-RBP is sufficient for the suppression of ORF57 promoter activation mediated by the viral transactivator RTA. Finally, we show that K-RBP inhibits RTA binding to ORF57 promoter. These findings suggest that the DNA-binding activity of K-RBP plays an important role in repressing viral promoter activity. Zhilong Yang http://digitalcommons.unl.edu/virologypub/196 http://digitalcommons.unl.edu/virologypub/196 Thu, 29 Oct 2009 12:16:57 PDT Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing. Feng Qu http://digitalcommons.unl.edu/virologypub/195 http://digitalcommons.unl.edu/virologypub/195 Wed, 28 Oct 2009 11:49:05 PDT Annotation of the 330-kb Chlorella virus PBCV-1 genome identified a 237 nucleotide gene (a438l) that codes for a protein with ~35% amino acid identity to glutaredoxins (Grx) found in other organisms. The PBCV-1 protein resembles classical Grxs in both size (9 kDa) and location of the active site (N-terminus). However, the PBCV-1 Grx is unusual because it contains a monothiol active site (CPYS) rather than the typical dithiol active site (CPYC). To examine this unique active site, four sitespecific mutants (CPYC, CPYA, SPYC, and SPYS) were constructed to determine if the N-terminal cysteine is necessary for enzyme activity. Wild type and both mutants containing N-terminal cysteines catalyzed the reduction of disulfides in a coupled system with GSH, NADPH, and glutathione reductase. However, both mutants with an altered N-terminal cysteine were inactive. The grx gene is common in the Chlorella viruses. Molecular phylogenetic analyses of the PBCV-1 enzyme support its relatedness to those from other Chlorella viruses and yet demonstrate the divergence of the Grx molecule. Lisa A. Fitzgerald http://digitalcommons.unl.edu/virologypub/194 http://digitalcommons.unl.edu/virologypub/194 Fri, 31 Jul 2009 08:03:29 PDT 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 or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip® assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes. Yoshimi Enose http://digitalcommons.unl.edu/virologypub/193 http://digitalcommons.unl.edu/virologypub/193 Fri, 19 Jun 2009 13:16:56 PDT Human herpesvirus (HHV) type 8 bas been detected in both classical and AIDS-related Kaposi's sarcoma, body-cavity lymphomas, and other types of tumors. HHV-8 has also been detected in DNA from peripheral blood mononuclear cells (PBMC) of some Kaposi's sarcoma patients and more readily in B cell fractions derived from panned cell subpopulations. Two patients were followed using several methods: in situ hybridization, solution-based polymerase chain reaction (PCR), and in situ PCR. HHV-8 was intermittently detected in plasma and detection correlated with detection in PBMC. In situ PCR demonstrated HHV-8 sequences in both peripheral blood B lymphocytes and, to a lesser extent, T lymphocytes. HHV-8 may undergo periods of viremia while at other times it is undetectable and infects circulating B cells and some T cells. William J. Harrington http://digitalcommons.unl.edu/virologypub/192 http://digitalcommons.unl.edu/virologypub/192 Fri, 19 Jun 2009 10:42:53 PDT Kaposi's sarcoma occurs at high incidence among Zambian adults and children, but there is a paucity of data on human herpesvirus 8 (HHV-8) incidence and routes of infection, especially in children. Between 1998 and 2004, the authors conducted a prospective study of viral transmission in a cohort of 684 children in Lusaka, Zambia, to estimate the annual incidence of HHV-8 from birth through 48 months of age. Maternal and pediatric human immunodeficiency virus type 1 (HIV-1) infection status was also determined. The results, based on 1,532 child-years of follow-up, showed that HHV-8 seroconversion occurs early in life. The incidence rate of HHV-8 seroconversion was 13.8 infections per 100 child-years by 48 months of age. HIV-1-infected children were at substantially higher risk for HHV-8 seroconversion (adjusted hazard ratio ¼ 4.60, 95% confidence interval: 2.93, 7.22). Maternal HIV-1 and HHV-8 infection status were not independently associated with risk of HHV-8 seroconversion in the child. HHV-8 antibody titers in children followed at all consecutive time points revealed seroreversion of HHV-8 antibodies, with undetectable titers in some children at one or more time points after seroconversion. These results demonstrate that cross-sectional serologic screening probably underestimates true HHV-8 seroprevalence in young Zambian children because of fluctuations in detectable antibody titers. Veenu Minhas http://digitalcommons.unl.edu/virologypub/191 http://digitalcommons.unl.edu/virologypub/191 Wed, 29 Apr 2009 12:53:49 PDT Background: The ProteomeLab PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial. Results: The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion: We offer some insight into the strengths and limitations of this emerging proteomic platform. Joshua Schlautman http://digitalcommons.unl.edu/virologypub/190 http://digitalcommons.unl.edu/virologypub/190 Wed, 29 Apr 2009 12:53:46 PDT Background: New, more sensitive and specific biomarkers are needed to support other means of clinical diagnosis of neurodegenerative disorders. Proteomics technology is widely used in discovering new biomarkers. There are several difficulties with in-depth analysis of human plasma/ serum, including that there is no one proteomic platform that can offer complete identification of differences in proteomic profiles. Another set of problems is associated with heterogeneity of human samples in addition intrinsic variability associated with every step of proteomic investigation. Validation is the very last step of proteomic investigation and it is very often difficult to validate potential biomarker with desired sensitivity and specificity. Even though it may be possible to validate a differentially expressed protein, it may not necessarily prove to be a valid diagnostic biomarker. Results: In the current study we report results of proteomic analysis of sera from HIV-infected individuals with or without cognitive impairment. Application of SELDI-TOF analysis followed by weak cation exchange chromatography and 1-dimensional electrophoresis led to discovery of gelsolin and prealbumin as differentially expressed proteins which were not detected in this cohort of samples when previously investigated by 2-dimensional electrophoresis with Difference Gel Electrophoresis technology. Conclusion: Validation using western-blot analysis led us to conclude that relative change of the levels of these proteins in one patient during a timeframe might be more informative, sensitive and specific than application of average level estimated based on an even larger cohort of patients. Jayme Wiederin http://digitalcommons.unl.edu/virologypub/189 http://digitalcommons.unl.edu/virologypub/189 Wed, 29 Apr 2009 12:43:15 PDT Background: PVs (PV) are small, non-enveloped, double-stranded DNA viruses that have been identified as the primary etiological agent for cervical cancer and their potential for malignant transformation in mucosal tissue has a large impact on public health. The PV family Papillomaviridae is organized into multiple genus based on sequential parsimony, host range, tissue tropism, and histology. We focused this analysis on the late gene products, major (L1) and minor (L2) capsid proteins from the family Papillomaviridae genus Alpha-papillomavirus. Alpha-PVs preferentially infect oral and anogenital mucosa of humans and primates with varied risk of oncogenic transformation. Development of evolutionary associations between PVs will likely provide novel information to assist in clarifying the currently elusive relationship between PV and its microenvironment (i.e., the single infected cell) and macro environment (i.e., the skin tissue). We attempt to identify the regions of the major capsid proteins as well as minor capsid proteins of alpha-papillomavirus that have been evolutionarily conserved, and define regions that are under constant selective pressure with respect to the entire family of viruses. Results: This analysis shows the loops of L1 are in fact the most variable regions among the alpha- PVs. We also identify regions of L2, involved in interaction with L1, as evolutionarily conserved among the members of alpha- PVs. Finally, a predicted three-dimensional model was generated to further elucidate probable aspects of the L1 and L2 interaction. John Lowe http://digitalcommons.unl.edu/virologypub/188 http://digitalcommons.unl.edu/virologypub/188 Fri, 27 Mar 2009 16:11:50 PDT HIV-1HXB2 5′LTR region, most of BIVR29 gag-pol segment and HIV-1HXB2 pol IN-3′LTR region were respectively amplified. A chimeric clone, designated as pHBIV3753, was constructed by cloning three fragments sequentially into pUC18. MT4 cells were transfected with pHBIV3753. The replication and expressions of the chimeric virus (HBIV3753) were monitored by RT activity and IFA. The results firstly demonstrated that it is possible to generate a new type of the BIV/HIV-1 chimeric virus containing BIV gag-pol gene. Guomin Chen