The Role of Hantavirus Glycoprotein Glycosylation on Infection of Vero E6 Cells Asra Khan* and Meda Higa Ph.D Department of Biological Sciences, York College of Pennsylvania Acknowledgments Literature Cited To determine the role of viral glycosylated glycoproteins in infection Objective  Hantaviruses belongs to the family Bunyaviridae and can cause Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS).  The viral membrane precursor is cleaved into two glycoproteins, G N and G C which play a vital role in virus-host interactions during infection. Both G N and G C contain putative N-linked or O-linked glycosylation sites.  Glycosylation aids in the process of protein folding and trafficking. Studies have shown that viral glycoproteins are critically involved in virus entry, however the mechanism of this interaction remains unclear (Shi and Elliott 2004; Zheng et al., 2007).  Due to the impact of Hantaviruses on human health, it is important to determine the mechanisms that contribute to infection including the role of glycosylation on viral glycoproteins. Zheng et al., 2007 Introduction Future Directions Glycan Competition Vero E6 cells treated with Mannose Vero E6 cells treated with DMEM Vero E6 cells treated with Glucose Infect Cells Infection Rate of Three Hantavirus Strains with Different Media Treatments Figure 3. VERO E6 cells were incubated with mannose, glucose or DMEM then media was removed. Infection was done with four different virus strains and analyzed. Compare glycosylation sites between viral strains and their relative levels of infection. Confirm viral deglycosylation using Western Blot Mutate putative glycosylation sites and assess differences in infection levels. Figure 1. A. HEK293T cells were transfected with viral plasmid. B. Vesicular Stomatitis Virus (VSV) core with Renilla Luciferase gene was used to infect HEK293T cells. C. Hantavirus pseudovirions were then used to infect VERO E6 cells and infection was analyzed by measuring light Discussion  The three treatment groups, treatment with the enzyme, mock treatment or spike treatment did not have any significant difference in infection rates. This may have been due to the use of the native form of the protein when protocol was conducted.  The use of a high mannose media, versus a glucose or DMEM media resulted in a significant decrease on infection rates. This suggests that there are high mannose binding receptors on all virus membranes needed for infection to take place. ANDV showed a higher infection rate compared to the others. This might have been due to the multiple glycosylation sites on its membrane including N-linked and O-linked glycosylation sites with high mannose glycan chains.  Conclusion: The experiments suggests that glycosylation aids in viral infection. Endo H protocol Deglycosylate with Endo H enzyme Virus Treated with enzyme Virus Mock Treated Virus Spiked with enzyme Infect Cells Figure 2. Viruses were incubated with enzyme for one hour and then infected onto VERO E6 cells. Treatment group contained enzyme, mock group contained no enzyme and spiked group had enzyme added to solution before spread on cells. I would like to thank Dr. Jeffery Thompson and Dr. Bridgette Hagerty for their assistance and advice throughout this project. I would like to thank the Department of Biological Sciences at York College of Pennsylvania for their support. Rate of Infection with Endo H Treatment for Three Hantavirus Strains Results Viral Plasmid Glycoprotein C.C. Pseudovirions LIGHT HEK 293T Cell Transfect VSV Infect with VSV VERO E6 Cell Infection with Pseudovirion A.A. B. Pseudovirions