The Role of The NS1 Protein in Zika Virus Pathogenesis Open Access
Habib, Rumi (Spring 2019)
Abstract
Zika Virus (ZIKV) is an emerging single-stranded positive-sense RNA virus of the genus Flavivirus. Most cases of ZIKV infection are mild or subclinical, resulting in an acute disease with common symptoms including a rash, fever, and joint pain. However, during the 2015-2016 ZIKV outbreak in South and Central America, it was observed that ZIKV infection can result in complications such as thrombocytopenia, Guillain–Barré syndrome, and most significantly, infant microcephaly. These observations showed that ZIKV pathogenesis is still poorly understood, leading the World Health Organization to declare the outbreak an international public health emergency in 2015 and list ZIKV as a priority pathogen. The ZIKV genome is divided into 3 structural and 7 non-structural proteins. In cells infected with ZIKV, non-structural protein 1 (NS1) dimerizes and plays a role in the viral replication complex. However, it has also been shown to be secreted from infected cells in a hexameric form (sNS1). This secreted form of the protein has been shown to be highly immunogenic and is being investigated as a potential vaccine candidate for ZIKV. This means that the immune response to sNS1 should act as a strong selective pressure against the secretion of the protein by the virus, yet sNS1 secretion is a strongly conserved feature of flavivirus pathogenesis, suggesting that sNS1 plays an important role in ZIKV-host interactions. In Dengue virus infection (a closely related virus), sNS1 has been implicated in the vascular leakage seen during the course of the disease, and in the generation of autoreactive antibodies. We hypothesized that sNS1 plays a similar part in the vascular permeabilization seen during ZIKV pathogenesis such as in the uterus, the blood-brain barrier, and the blood-ocular barrier. To test this hypothesis, we used a lentiviral vector to generate a cell line stably expressing ZIKV NS1. In addition, we injected mice with ZIKV NS1 to look for impacts on proteins involved with the coagulation pathway, and the generation of autoreactive antibodies.
Table of Contents
I. Introduction ...................................................................................................................... 1
The Impact of Zika Virus ................................................................................................. 1
The sNS1 Protein ............................................................................................................ 2
Investigating the Role of sNS1 in ZIKV Pathogenesis................................................... 4
II. Materials and Methods ................................................................................................... 5
Molecular Cloning ........................................................................................................... 5
Generation of a Stable Cell Line..................................................................................... 6
ELISA for NS1 Secretion ................................................................................................ 7
Western Blot for NS1 Secretion...................................................................................... 8
Mouse Antibody Responses ........................................................................................... 8
Anti-plasminogen Antibody ELISA.................................................................................. 9
III. Results ......................................................................................................................... 10
Generation of Stably Transduced Cell Line ................................................................. 10
Confirmation of NS1 Secretion ..................................................................................... 11
Mouse Antibody Responses to NS1............................................................................. 12
Generation of Anti-plasminogen Antibodies................................................................. 13
IV. Discussion ................................................................................................................... 14
V. Figures
Figure 1 ...................................................................................................................... 18
Figure 2 ...................................................................................................................... 19
Figure 3 ...................................................................................................................... 20
Figure 4 ...................................................................................................................... 21
Figure 5 ...................................................................................................................... 22
Figure 6 ...................................................................................................................... 23
Figure 7 ...................................................................................................................... 24
Figure 8 ...................................................................................................................... 25
VI. Citations ...................................................................................................................... 26
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