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Hendra Virus V Protein Inhibits Interferon Signaling by Preventing STAT1 and STAT2 Nuclear Accumulation Jason J. Rodriguez, Lin-Fa Wang, and Curt M. Horvath Presented by Morgan Wood What is Hendra Virus? • Also called equine morbillivirus. • It was first discovered in horse populations in Australia, with the only three human cases occuring during an outbreak in 1994 in Hendra, a suburb of Brisbane, Australia. • Both Hendra And Nipah Viruses are members of the family Paramyxoviridae. This family covers a large group of Negative-strand RNA viruses that cause Human and animal diseases. They are together the founding members of a genus, Henipavirus. • It is believed that both Hendra and Nipah viruses have natural reservoirs in large bat species in the areas where the viruses occur. Hendra Virus Cont. • The symptoms of Hendra virus include respiratory illness with sever flu-like symptoms, and can progress to encephalitis (swelling of the brain). • Both Hendra virus and Nipah virus have the potential to be fatal. • Both of these viruses infect humans when they come into contact with bodily fluids or secretions of infected animals; horses in the case of Hendra, and pigs in the case of Nipah. • Of the three cases of Hendra virus that have been reported, two died as a result of the infection and the third had a lengthy recovery. Interferon signaling • Interferon signaling is involved in the innate immune response to viruses in the body. • The signal is transmitted by proteins called STATs, that dimerize, once activated, and are then able to travel into the nucleus and drive transcription of immune genes. • Preventing nuclear accumulation of STAT proteins is a common way that this signal pathway is disrupted. Questions 1. Are the V proteins of Hendra and Nipah Viruses Similar? 2. Does the Hendra virus V protein interfere with interferon signaling? 3. Does the Hendra virus V protein form aggregates with the STAT1 & STAT2 proteins? 4. Does the Hendra virus V protein prevent the nuclear accumulation of STAT1 & STAT2 proteins? Methods • Amino acid sequencing was used to determine protein sequences. • Human fibrosarcoma 2fTGH cells were used in the Luciferase assay as well as in the immunostaining assay. • 293T cells were used in the V protein aggregation experiments as well as in the Luciferase assay experiments, but the data was not shown in the latter. • Green flourescent protein, from the sigma corporation, was used in an experiment as well. • Western blot was done to determine formation of an aggregate. Methods Cont. • Chromatography was used to separate out molecular complexes of different sizes for fig. 2b. • A solution is passed through a seperating column, fractions of the solution are collected seperatly as they come out of the column. These fractions contain different sized complexes in them. • Larger things move through column faster, while small molecules are trapped in pores in side the filter media, they end up being washed out in later fractions. • The molecules in the fractions are then blown apart to allow for easier electrophoresis, and are then run through a page process. Are the V proteins of Hendra and Nipah Viruses Similar? • In order to establish the relationship between Hendra and Nipah viruses, the authors sequenced the amino acids of the V proteins for both species. • The overall similarity was 58%. There was an 81% similarity between aa 1140, 44% between 141405, and 83% between 406-457. • Answer: Yes, they are quite similar. Does the Hendra virus V protein interfere with interferon signaling? • It has been established in previous studies that Nipah virus V protein interferes with interferon signaling, so it will be used as the standard to judge Hendra virus against. • Two luciferase reporter construct were assembled and transfected into human fibrosarcoma 2fTGH cells, and 293 T cells. • Cells were either transfected with HA-tagged Nipah virus V (NiV), Hendra virus V (HeV), or HAtagged vector with no cDNA. • They were then treated with (+) or without (-) IFN 12 h prior to lysis. • Answer: yes, it interferes with signaling Does the Hendra virus V protein form aggregates with the STAT1 & STAT2 proteins? • 293T cells were transfected with either blank expression vectors (C), Flagtagged Hendra virus V expression vectors (H), or Flag-tagged Nipah virus V expression vectors (N). • The left column shows basal levels of STAT proteins as well as the Flag protein in the two right lanes. • The right column shows a coimmunoprecipitation of the STAT & V protein complexes. Does the Hendra virus V protein form aggregates with the STAT1 & STAT2 proteins? • 293T cells were transfected with either green flourescent protein (GFP), NiV, or HeV. • Cells were then lysed and the solution run through a chromatography seperator. • Fractions from each type of lysate were then subjected to a western blot using either STAT1 or STAT2 antibodies. • The lower the fraction number that the STAT protein shows up in, the larger the complex that went through the column. • The aggregates formed were about 500kDa in size. • Answer: YES. Does the Hendra virus V protein prevent the nuclear accumulation of STAT1 & STAT2 proteins? • Immunostaining was used to show that Hendra virus V protein actually inhibits the nuclear accumulation of the STAT1 & STAT2 proteins. • Human fibrosarcoma 2fTGH cells were transfected with plasmid encoding HA-tagged Hendra virus V protein. • Cells were either left untreated (UNT) or treated with IFNg for 30 minutes prior to fixation. • The cells were stained with a red flourescent stain for the HA tag, and a green flourescent stain for either STAT1 or STAT 2. • Cells without the HeV appear with green stain but no red stain. • Answer: Yes. Questions 1. 2. 3. 4. Are the V proteins of Hendra and Nipah Viruses Similar? Does the Hendra virus V protein interfere with interferon signaling? Does the Hendra virus V protein form aggregates with the STAT1 & STAT2 proteins? Does the Hendra virus V protein prevent the nuclear accumulation of STAT1 & STAT2 proteins? • Yes, 58% overall. • Yes, reducing activity by 80%. • Yes, about 500kDa in size. • Yes. Conclusions • Hendra virus and Nipah virus are very closely related, and their V proteins inhibit interferon signaling in very similar ways. • While human infections of these diseases remain fairly low, a good understanding of them is helpful, since both are zoonotic diseases (passed from animals to humans). • Understanding of these diseases could lead to breakthroughs in the treatment of a number of illnesses that are common in the zoo field. Refferences • Rodriguez, J. J., Wang, L., Horvath, C.M. Hendra virus V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation. Journal of Virology 77(21): Nov. 2003 p. 11842-11845. • Center for Disease Control website www.cdc.org • Dr. Bulla’s website