Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

Human immunodeficiency virus type 1 reverse transcriptase: enhancement of activity by interaction with cellular topoisomerase I.

A number of studies have suggested that topoisomerase I (topo I) activity may be important in human immunodeficiency virus type 1 (HIV-1) replication. Specifically it has been reported that purified virus particles have topo I activity and that inhibitors of this enzyme can inhibit virus replication in vitro. We have investigated a possible association of HIV-1 gag proteins with topo I activity. We found that whereas the gag-encoded proteins by themselves do not have activity, the nucleocapsid protein p15 can interact with and enhance the activity of cellular topo I. Furthermore it could be demonstrated that topo I markedly enhanced HIV-1 reverse transcriptase activity in vitro and that this could be inhibited by the topo I-specific inhibitor camptothecin. The findings suggest that cellular topo I plays an important role in the reverse transcription of HIV-1 RNA and that the recruitment of this enzyme may be an important step in virus replication.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.

Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.

Study of the role of retinoblastoma protein in terminal differentiation of murine erythroleukemia cells.

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle.

Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Thesis (Ph.D.)--University of Washington, 2016-06

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Selection pressure-driven evolution of the Epstein-Barr virus-encoded oncogene LMP1 in virus isolates from southeast Asia

The geographically constrained distribution of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in southeast Asian populations suggests that both viral and host genetics may influence disease risk. Although susceptibility loci have been mapped within the human genome, the role of viral genetics in the focal distribution of NPC remains an enigma. Here we report a molecular phylogenetic analysis of an NPC-associated viral oncogene, LMP1, in a large panel of EBV isolates from southeast Asia and from Papua New Guinea, Africa, and Australia, regions of the world where NPC is and is not endemic, respectively. This analysis revealed that LMP1 sequences show a distinct geographic structure, indicating that the southeast Asian isolates have evolved as a lineage distinct from those of Papua New Guinea, African, and Australian isolates. Furthermore, a likelihood ratio test revealed that the C termini of the LMP1 sequences of the southeast Asian lineage are under significant positive selection pressure, particularly at some sites within the C-terminal activator regions. We also present evidence that although the N terminus and transmembrane region of LMP1 have undergone recombination, the C-terminal region of the gene has evolved without any history of recombination. Based on these observations, we speculate that selection pressure may be driving the LMP1 sequences in virus isolates from southeast Asia towards a more malignant phenotype, thereby influencing the endemic distribution of NPC in this region.

Epstein-Barr virus-associated Hodgkin's lymphoma

Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30–50% of HL cases are Epstein–Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus’ role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy.

Heterologous RNA encapsidated in pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Angstrom resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.