Functional Studies of Vesicular Stomatitis Virus Matrix Protein and its
Modulation of NF-κB
Summer 2009 Proposal
Christopher Ried
ABSTRACT
Vesicular stomatitis virus (VSV) has been used as a model system to understand the modulation of
interferon (IFN), an important component of innate immunity that helps cells block replication of a
virus. IFN is activated through detection of viral dsRNA, which triggers multiple signaling cascades.
One of these cascades activates NF-κB, a cellular transcription factor that binds to the IFN gene to
induce expression of the IFN gene. The goal of this project is to determine if the VSV matrix (M)
protein suppresses activation of NF-κB. Importantly, development of the c-myc/M fusion protein
will allow us to prove expression of the VSV M protein, something that has hindered our research
for quite a while. These functional studies will provide evidence that will provide a deeper
understanding how the M-protein regulates NF-κB activation and therefore IFN gene expression.
Since VSV is currently being developed as an innovative type of cancer therapy, these studies could
lead to development of safer and more effective treatments for many types of cancers.
BACKGROUND AND SIGNIFICANCE
VSV is a member of the Rhabdoviridae family. This virus is a model system used for many
aspects of virology and cell biology (regulation of transcription, translation, gene regulation, etc). Its
negative sense RNA genome consists of five genes that encode for five multifunction proteins (N, P,
M, G and L) (Wagner and Rose, 1996). This virus can kill a host very quickly, in part because of its
inhibitory effect on host macromolecular synthesis and the cellular antiviral IFN response (Marcus
and Sekellick, 1985).
Research in our laboratory focuses on gaining an improved understanding of how VSV
regulates the IFN gene. In fact, the VSV model system was used to discover that the IFN gene is
inducible, and many aspects of gene regulation have been discovered by understanding how VSV
affects this highly regulated gene (Marcus and Sekellick, 1985). Several signal transduction cascades
are activated upon viral entry into a cell, leading to activation of three major transcription factors
necessary for induction of the IFN gene. These include the AP-1 complex, NF-κB and IFN
Regulatory Factor 3 (IRF-3) (Wathelet et al., 1998). Upon activation these transcription factors move
from the cytoplasm to the nucleus where they bind to the IFN promoter and trigger transcription of
IFN mRNA (Bonjardim et. al, 2005). The newly translated IFN protein is secreted from the cell
allowing it to bind to virus infected and non-infected neighbor cells to initiate ~200 genes that
interfere with virus replication (reviewed by Samuel in 1991 and Sen in 1992) and produce more
IFN (Samuel, 1991).
NF-κB is a transcription factor that is important for regulation of multiple important cellular
events including inflammation and the IFN antiviral response. Normally NF-κB is present in the
cytoplasm where it is unable to drive transcription of multiple genes because it is tightly bound to
IκB-α, an inhibitory protein which prevents NF-κB from moving into the nucleus. (Karin et. al,
2000) However under viral intrusion, NF-κB is activated, which means it relocates to the nucleus
where it can bind DNA and summon other proteins necessary to induce the IFN gene.
Previous work done in our laboratory suggests that VSV blocks NF-κB activation, and this
may in part explain how VSV suppresses IFN gene induction. Recent findings suggest that the VSV
M protein may be responsible for this regulation. In addition, the M protein is known to inhibit
general host transcription, therefore this protein might have multiple functions that limit
transcription of the IFN gene. It appears however that the story may be even more complicated!
Arthur Totten’s recent findings suggest that the M gene alone is not sufficient to suppress IFN.
These results suggest VSV may regulate the IFN gene by other pathways than regulation of NF-κB
activation and inhibition of general host transcription.
Gaining a better understanding of how the IFN gene is regulated in VSV-infected cells will
further increase our understanding of how this very important model system is regulated. We believe
this knowledge will improve our overall understanding of how eukaryotic genes can be induced
and/or suppressed. Our work may also help develop VSV into a safer and more effective treatment
for many different types of cancer.
PROJECT DESIGN AND GOALS
Our previous findings suggest that the M protein alone is sufficient to block NF-κB
activation, and we hope to confirm and expand upon these findings. To accomplish this goal, an
expression vector encoding a c-myc/M fusion protein will be generated so we can verify expression
of the M protein in transfected cells (Specific Aim 1) and methods to detect this fusion protein will
be developed (Specific Aim 2). We will also further define the VSV proteins involved in this
regulation (Specific Aim 3). We will determine if the c-myc M protein alone is sufficient for
regulation of NF-κB (Objective 3.1), or if another VSV gene, or combination of these genes,
regulate NF-κB (Objective 3.2 and 3.3).
Specific Aim 1 – Generate a c-myc/M expression plasmid using a “sticky end” PCR protocol
(Walker et. al. 2008).
Objective 1.1
Objective 1.2
Objective 1.3
Objective 1.4
Infect cells and purify total RNA
Amplify the M gene from wild type and T1026R1 VSV using a “sticky end” PCR
protocol.
Ligate the PCR fragment into the pcDNA3.1 expression plasmid
Verify the clone by restriction enzyme digestion and sequencing, and produce large
quantities of transfection-grade plasmid.
Goal: Our previous attempts to produce functional GFP-VSV fusion proteins failed,
presumably because the relatively large GFP protein caused the fusion protein to fold improperly.
We will now generate recombinant eukaryotic expression vectors, encoding a c-myc/M fusion gene
downstream of a heterologous (CMV) promoter. A c-myc epitope tag (EQKLISEEDL) (Lin, et. al.
1994) will be added so expression of these proteins can be easily verified (Specific Aim 2). The cmyc epitope is very small in comparison to GFP, so we hope that the c-myc/M fusion protein will be
functional.
Experimental design: Nuclear and cytoplasmic RNA will be isolated from VSV-infected
cells using the Ambion® RNAqueous-for-PCR kit according to the manufacturer’s instructions. The
RNA will be quantitated and its integrity verified by agarose gel electrophoresis. RT-PCR is then
used to amplify the VSV M gene. The primers for the “sticky end cloning” have already been
designed and ordered. First-strand cDNA will be synthesized using the ImProm-II Reverse
Transcription System (Promega). During the PCR, DeepVent DNA polymerase will be used because
it contains a proofreading function, minimizing the chance that errors are introduced into the VSV
genes during amplification. The PCR products will then cloned into pCDNA3.1, a constitutive
eukaryotic expression plasmid, according to the manufacturer’s instructions (Invitrogen). Colonies
will be screened by restriction enzyme digestion and sequencing. Once identified, large quantities of
the correct clones will be generated using Qiagen Maxiprep kits. These expression vectors will be
useful reagents for this proposal and many future projects. Students in the lab do have experience
with cloning, however the “sticky end cloning” is a new approach.
Specific Aim 2 – Confirm expression of the c-myc/M fusion protein in transfected cells.
Objective 2.1
Verify expression of the c-myc/M fusion proteins by Western blot analysis.
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Objective 2.2
Verify expression and localization of the c-myc/M fusion proteins by
immunofluorescence.
Goal: Confirming expression of the VSV M protein in transfected cells is not simple since the
protein inhibits its own synthesis along with inhibition of general host transcription and translation.
Compounding this difficult, there are no efficient antibodies available to detect the VSV M protein.
The c-myc/M expression vector proposed in Specific Aim 1 will allow us to confirm expression of
the M protein using a primary antibody that recognizes the c-myc epitope. Expression of the c-mycM fusion protein will be confirmed via Western blotting and immunofluorescence.
Experimental design: Briefly, Western blotting involves collection of total cell lysates from
transfected cells, quantification of total protein (Bradford assay), separation of proteins by their
molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer
of the proteins to a nitrocellulose membrane. The membrane is then exposed to a primary antibody
that will recognize the c-myc epitope. Following several washes, the membrane is then exposed to a
second antibody that is conjugated to horse radish peroxidase, which allows visualization via
chemiluminescence. Immunofluorescence will be used in Objective 2.1. Cells will be transfected and
grown on clean, sterile coverslips. After fixing (4% paraformaldehyde in PBS) and permeabilizing
(0.1% triton X-100 in PBS), the cells will be covered with a primary antibody that recognizes the cmyc epitope. After many washings a secondary antibody solution that will recognize the primary
antibody, and that is conjugated to a fluorescent probe (FITC) will be placed on the cells. The cells
will be washed, mounted, and observed using a fluorescent microscope. Images can be captured to
be used in figures. Both of the techniques have been performed in the laboratory, although not for
quite a while.
Specific Aim 3 – Identify the viral component(s) responsible for regulation of NF-κB Activation.
Objective 3.1
Confirm that the VSV M protein alone is (or is not) sufficient for NF-κB activation
using our new c-myc/M construct (TransAM and co-immunofluorescence)..
Objective 3.2
Examine NF-κB activation in cells transfected with a G, L, N or P expression
plasmid
Examine NF-κB activation in cells co-transfected with the c-myc/M fusion protein
in combination with the other VSV proteins
Objective 3.3
Goal: Our preliminary results suggest that the VSV M protein alone is able to suppress NFκB regulation. We hope to confirm and expand upon these results by first determining what effect of
the M protein expression has on NF-κB suppression. We will then determine the effect of each of the
VSV proteins alone, or in combination, on NF-κB regulation.
Experimental design: Cells will be transfected using the Nucleofector technology
(electroporation-based transfection). Approximately 48 hours post-transfection, the cells will be
infected with T1026R1 to induce IFN production. We will then determine if NF-κB has been
activated using the TransAM assay and co-immunofluorescence. I have been using the TransAM
assay extensively, which allows quantitation of NF-κB activation in nuclear lysates (it is similar to
an ELISA assay). Co-immunofluorescense is similar to the immunofluorescence assay described
above, however we will use one primary antibody to detect the viral protein and a second primary
antibody to detect NF-κB. Other students have used this method in the past, however it will be new
to me.
REFERENCES
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Bonjardim, Caudio A., et. al. 2005. Interferons (IFNs) are key cytokines in both innate and
adaptive antiviral immune responses-and viruses counteract IFN action. Microbes and Infection 7:
569-578.
Lin, Chao, et. al. 1994. Processing in the Hepatitis C Virus E2-NS2 Region: Identification of p7
and Two Distinct E2-Specific Products with Different C Termini.
Virology 68: 5063-5073.
Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18:621–663.
Marcus, P. I., and M. J. Sekellick. 1985. Interferon induction by viruses. XIII. Detection and assay
of interferon induction-suppressing particles. Virology 142:411-415.
Samuel, C. E. 1991. Antiviral actions of interferon. Interferon-regulated cellular proteins and their
surprising selective antiviral activities. Virology 183:1-11.
Sen, R., and P. Lengyel. 1992. The interferon system. A bird's eye view of its biochemistry. J. Biol.
Chem. 267:5017-5020.
Wagner, R. R., and J. K. Rose. Rhabdoviridae: 1996. The viruses and their replication 3rd ed. in
"Fundamental Virology" (B. N. Fields, D. M. Knipe, and P. M. Howley, Eds 561-604. LippincottRaven, Philadelphia
Walker, Andrew, et. al. 2008. A method for generating sticky-end PCR products which facilitates
unidirectional cloning and the one-step assembly of complex DNA contructs. Plasmid 59: 155-162.
Wathelet, M. G., C. H. Lin, B. S. Parekh, L. V. Ronco, P. M. Howley, and T. Maniatis. 1998.
Virus infection induces the assembly of coordinately activated transcription factors on the IFN-b
enhancer in vivo. Molecular Cell 1:507-518.
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