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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 CitedHiga, M. M., Petersen, J., Hooper, J., and Doms, R. W. 2011. Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies. Virology. 423:134-142. Shi, X. and Elliott, R. M., 2004. Analysis of N-Linked Glycosylation of Hantaan Virus
Glycoproteins and the Role of Oligosaccharide Side Chains in Protein Folding and Intracellular Trafficking. Journal of Virology. 78: 5414–5422.
Zheng, F., Ma, L., Shao, L., Wang, G., Chen, F., Zhang, Y., and Yan, S. 2007. Defining the N- Linked Glycosylation Site of Hantaan Virus Envelope Glycoproteins Essential for Cell Fusion. Journal of Microbiology. 45:41-47.
• 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, GN and GC which
play a vital role in virus-host interactions during infection. Both GN and GC 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 DirectionsGlycan 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 protocolDeglycosylate 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.
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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.
Pseudovirions
LIGHT
HEK 293T Cell
Transfect
VSV
Infect with VSV
VERO E6 Cell
Infection with Pseudovirion
A.
B.
Pseudovirions
Figure 4. Infection rate of three virus strains (HNTV, PUUV, ANDV) with Endo H activity in three different treatment groups (treated, mock, spike). RLU represents Renilla Luciferase Units. A two-way ANOVA was conducted to evaluate the means between the three treatment groups ( = 0.066, p>0.05) and the means among the virus strains ( = 2.595, p=0.053). A multiple comparison test was performed to analyze the differences between the treatments for each viral strain. There was a significant difference between PUUV mock treated and PUUV spiked. The error bars show the standard error of means ± (SEM).
Figure 5. Infection rate of three different virus strains (HNTV, PUUV, ANDV) in three different media types (Mannose, Glucose, and DMEM). RLU represents Renilla Luciferase Units. A two-way ANOVA was conducted to evaluate the significant means between the three treatment groups ( =2, p=0.0251) and the differences between the viruses ( 25.17, p=0.008). A multiple comparison test was performed to analyze the differences between the treatments and viral strains. Specifically, there was a significant difference between the mannose and the glucose group. There was a significant difference between the mannose treated ANDV and the glucose treated ANDV. The error bars show the standard error of means (SEM).
