1. 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

2. 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.

3. 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.

4. 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.

5. 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?

6. 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.

7. 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.

8. 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 1- 140, 44% between 141- 405, and 83% between 406-457. Answer: Yes, they are quite similar.

9. 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 HA- tagged vector with no cDNA. They were then treated with (+) or without (-) IFN 12 h prior to lysis. Answer: yes, it interferes with signaling

10. Does the Hendra virus V protein form aggregates with the STAT1 & STAT2 proteins? 293T cells were transfected with either blank expression vectors (C), Flag- tagged 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.

11. 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.

12. 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 IFN  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.

13. 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? Yes, 58% overall. Yes, reducing activity by 80%. Yes, about 500kDa in size. Yes.

14. 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.

15. 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 www.cdc.org Dr. Bulla’s website