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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
Introduction
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.
Endo H protocol Virus Treated with enzyme Deglycosylate with Endo H enzyme Virus Mock Treated Virus Spiked with enzyme Results Rate of Infection with Endo H Treatment for Three Hantavirus Strains
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.
Zheng et al., 2007
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. 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 ( F (2,24) the means among the virus strains ( F (3,24) = 0.066, p>0.05) and = 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).
Conclusion: The experiments suggests that glycosylation aids in viral infection.
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Objective
To determine the role of viral glycosylated glycoproteins in infection
Glycan Competition Vero E6 cells treated with
Mannose
Vero E6 cells treated with
Glucose
Vero E6 cells treated with
DMEM
Infection Rate of Three Hantavirus Strains with Different Media Treatments
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Future Directions Compare glycosylation sites between viral strains and their relative levels of infection.
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Confirm viral deglycosylation using Western Blot
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Mutate putative glycosylation sites and assess differences in infection levels.
Pseudovirions
Glycoprotein Viral Plasmid
Transfect
VSV
Infect with VSV
HEK 293T Cell
Literature Cited
Higa, 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.
C
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Infection with Pseudovirion
VERO E6 Cell
LIGHT
Infect Cells Pseudovirions
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 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. 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 ( F (2,24) =2, p=0.0251) and the differences between the viruses ( F (3,24) 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).
Acknowledgments
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.