Download Caco-2 cell culture and DNA transfection

Genome-wide Analysis of Gene Expression Profiles Induced by Norwalk
Virus Capsid Protein Gene
P8-4
P. Liu1. YH. Li2, H. Hsiao1, N. Wagar2, C. L Moe1
1Rollins School of Public Health, 2Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322
Noroviruses (NoV) are the leading cause of outbreaks of non-bacterial
gastroenteritis both in the US and worldwide. Currently, a vaccine is not available,
but several vaccine strategies target the norovirus capsid protein encoded by the
open reading frame 2 (ORF-2). Recombinant norovirus capsid protein can selfassemble into virus-like particles (VLP) that are antigenically and morphologically
similar to native whole norovirus. In order to better understand the molecular
mechanism of ORF-2 as a norovirus vaccine candidate, we transfected Norwalk
virus ORF-2 plasmid DNA into a human intestinal cell line Caco-2. Analysis by
SYBR green real-time PCR showed that ORF2 expression increased
5,039~27,364-fold in the ORF2-transfected cells compared to cells transfected with
only the vector. The whole genome expression profiles were analyzed in triplicate
experiments using an Affymetrix human U133_ 2.0 array representing
approximately 47,000 transcripts. A total of 3,483 transcripts exhibited at least 1.5fold differences in their expression levels. Statistical analyses indicated that 54
transcripts were significantly different (p<0.05) between ORF2-transfected and
vector only-transfected cells. Out of these, 28 mRNAs were up-regulated while 26
mRNAs were down-regulated. Hierarchical clustering indicated that the gene
expression patterns of ORF2- and vector-transfected cells could be clearly
distinguished. We found that zinc finger protein 236 (ZNF236), zinc finger protein
519 (ZNF519) and B-cell CLL/lymphoma 10 (BCL10) were the most important upregulated genes in this study. Signal pathway analysis showed that the zinc finger
proteins are involved in transcriptional regulation and that BCL10 plays a key role
in antigen-receptor-mediated lymphocyte activation and the development of B and
T cells. ORF2 transfection resulted in the down-regulation of chemokine receptor 1
(CCR1) and tumor necrosis factor superfamily member 8 (TNFSM8), both genes
involved in innate and adaptive immune responses. These results provide novel
information on the complex immune response at the transcriptional level in human
intestinal cells induced by a norovirus vaccine candidate antigen.
Microarray analysis
The Affymetrix CEL files were imported into GeneSpring 7.3 and normalized by
GC-Robust Multichip Average (GCRMA). Firstly we removed genes with absent
signal, and filtered out genes with present or marginal signal and with at least
two-fold increased or decreased mRNA expression between ORF2-transfected
and vector only transfected cells. Genes with statistically different expression
between ORF2-transfected and vector only transfected cells (p<0.05) were
identified by one way ANOVA analysis. Average-linkage hierarchical clustering
was carried out to cluster the distinct patterns in gene expression and the
relationships between the samples. Genes identified by GeneSpring were
uploaded into the Ingenuity Pathways Analysis tool to further analyze the gene
ontology, including biological processes, cellular components, molecular
functions and genetic networks.
RESULTS
ORF2
8
Vector
Noroviruses (NoV), belong to the family of caliciviridae, are the leading cause of
outbreaks of non-bacterial gastroenteritis both in the U.S. and worldwide.
Currently, a vaccine is not available but certain high-risk groups, e.g. food
handlers, health care workers, the military and probably the old people, are in a
highly demand of norovirus vaccine. NoV genome is divided into three open
reading frames (ORFs), ORF1, ORF2 and ORF3, in which ORF2 encodes
norovirus capsid protein. Expression of ORF2 in baculovirus [1,2], Venezuelan
equine encephalitis virus (VEE) [3,4] and Yeast [5], respectively, is capable to form
virus-like particles (VLPs) that are antigenically and morphologically similar to
native whole norovirus but without viral RNA, and both monovalent and multivalent
VLPs could elicit systemic and mucosal immune response in human volunteers [6]
and in mice [7]. Furthermore, one study observed a Th1/Th2-like cellular response
in mice induced by genogroup II norovirus VLPs [8]. In order to better understand
the importance of ORF2 as norovirus vaccine candidate, we transfected Norwalk
virus ORF2 plasmid DNA into a human mammalian intestinal cell line Caco-2. The
whole genome expression profiles in ORF2 and vector alone-transfected cells
were compared in triplicate experiments using an Affymetrix human U133_ 2.0
array.
• ORF2 plasmid DNA transfection into human mammalian Caco-2 cells resulted
in a significant ORF2 mRNA expression from triplicate experiments.
2
• A total of 761 transcripts exhibited at least 2-fold differences and 54 transcripts
were statistically significant different between ORF2-transfected and vector
only-transfected cells.
0
1st
2nd
3rd
Fig. 1. ORF2 expression in Caco-2
mammalian cells with triplicate experiments.
SYBR green real-time PCR showed that
ORF2 expression increased 104-105.8-fold
in the ORF2-transfected Caco-2 cells
compared to the vector alone transfected
cells.
Fig. 2. Hierarchical clustering of 134 genes
with statistical significant different expression
between ORF2 transfected cells and vector
only transfected cells. Each row represents a
single gene; each column represents either
ORF2 transfected or vector transfected sample;
red color is up-regulated genes and green is
down-regulated genes in ORF2 transfected
versus vector transfected cells
Fig. 3. Pathways that 2-fold changed genes
were involved in. The x-axis displays pathways
that 2-fold altered genes involved in; Y-axis
displays the ratio of genes in a given pathway
that were 2-fold change divided by total
numbers of genes that make up that pathway.
METHODS
ORF2 Clone
We obtained a gift NV ORF2 clone that ORF2 fragment was cloned into a
pSPORT expression vector, and also a blank vector from Dr. Kim Green at NIH.
Both ORF2 and pSPORT plasmid DNAs were transformed to E.coli and the DNA
was confirmed by endonuclease digestion.
Caco-2 cell culture and DNA transfection
A human intestinal cell monolayer (Caco-2) was used as our in vitro mode of
transfection with the NV ORF2 expression vector and pSPORT vector. Caco-2
cells (ATCC, VA) was grown in Dulbecco's Modified Eagle's Medium
supplemented with 1 % nonessential amino acids, 10 % fetal calf serum (FCS),
1000 U/liter penicillin, and 1 mg/liter streptomycin. NV ORF2 and pSPORT vector
DNA were transfected to Caco-2 cells using lipofectamine 2000 reagent on three
separate days. RNA was extracted from the transfected cells
Total TNA extraction and cRNA synthesis
Total RNAs from Caco-2 cells were prepared with the RNeasy Mini kit (Qiagen Inc,
CA). and their integrities were verified on an Agilent Bioanalyzer. First-strand
cDNAs were synthesized from 5 µg of each RNA sample. After RNase H-mediated
second-strand cDNA synthesis, double-stranded cDNAs were purified and
biotinylated complementary RNAs (cRNAs) were generated by an in vitro
transcription. Biotinylated cRNAs were cleaned up, fragmented, and hybridized to
Affymetrix Human Genome U133 Plus 2.0 Array chips. Chips were processed
using an Affymetrix fluidics station and scanned on an Affymetrix scanner 3000
with workstation. Images were processed and raw data were extracted with
GeneChip Operating Software (GCOS).
Fig. 7. 2-fold changed genes involved in the
Immune response. Red are upregulated
(ORF2 vs. vector); Green are downregulated
genes; the intensity of color indicates the
degree of down or upregulation;
cytokine;
enzyme;
transcription regulatior;
transporter.
SUMMARY & CONCLUSIONS
4
NV ORF2 transfection
INTRODUCTION
Fig. 6. Genes with 2-fold altered expression
involved in the gastrointestinal diseases.
Red are upregulated (ORF2 vs. vector);
Green are downregulated genes; the intensity
of color indicates the degree of down or
upregulation;
cytokine;
enzyme;
transcription regulatior;
transporter.
NV ORF2 quantitation
6
Log10 of NV ORF2 RNA titiers
ABSTRACT
Pengbo Liu, Ph.D.
Rollins School of Public Health
Emory University
1518 Clifton Road, L36
Atlanta, GA 30322
Phone: (404)727-9218
Email: [email protected]
• Hierarchical clustering analysis could clearly divide the expression profiles of
ORF2 and vector alone transfected samples into two distinct groups, while could
also distinguish the 134 statistically significant different genes into various clusters.
• ORF2 transfection resulted in numbers of 2-fold changed genes that are involved
in several immunological pathways, e.g. antigen presentation pathway, interferon
signaling, IGF-1(insulin-like growth factor 1) signaling, etc.
• FUT4 (fucosyltransferase 4), FUT1, TNFSF15 (tumor necrosis factor superfamily
member 15), SLC4A4 (solute carrier family 4, member 4) are 2-fold changed genes
that involved in the gastrointestinal diseases.
ACKNOWLEDGEMENT
This study was supported by Emory University Research Committee (URC). We
are grateful to Dr. Kim Green at NIH for her kind gift of the Norwalk virus ORF2
clone.
REFERENCES
Fig. 4. Genes with 2-fold altered expression
participated in the antigen presentation pathway.
Genes in red are upregulated (ORF2 vs. vector);
green are downregulated genes; represents
transmembrane receptor;
represents
transcription regulation.
Fig. 5. Genes with 2-fold altered expression
participated in the pathway of leukocyte
extravasation signaling. Genes in red are
upregulated (ORF2 vs. vector); and the green
are downregulated genes;
enzyme;
group
or complex;
kinase;
other.
1. Jiang X, Wang M, Graham DY, Estes MK: Expression, self-assembly, and antigenicity of the
norwalk virus capsid protein. Journal of Virology 1992;66:6527-6532.
2. Green KY, Kapikian AZ, Valdesuso J, Sosnovtsev S, Treanor JJ, Lew JF: Expression and selfassembly of recombinant capsid protein from the antigenically distinct hawaii human calicivirus.
Journal of Clinical Microbiology 1997;35:1909-1914.
3. Baric RS, Yount B, Lindesmith L, Harrington PR, Greene SR, Tseng FC, Davis N, Johnston RE,
Klapper DG, Moe CL: Expression and self-assembly of norwalk virus capsid protein from
venezuelan equine encephalitis virus replicons. Journal of Virology 2002;76:3023-3030.
4. Harrington PR, Yount B, Johnston RE, Davis N, Moe C, Baric RS: Systemic, mucosal, and
heterotypic immune induction in mice inoculated with venezuelan equine encephalitis replicons
expressing norwalk virus-like particles. Journal of Virology 2002;76:730-742.
5. Xia M, Farkas T, Jiang X: Norovirus capsid protein expressed in yeast forms virus-like particles
and stimulates systemic and mucosal immunity in mice following an oral administration of raw
yeast extracts. Journal of Medical Virology 2007;79:74-83.
6. Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Estes MK: Humoral, mucosal, and
cellular immune responses to oral norwalk virus-like particles in volunteers. Clinical
Immunology 2003;108:241-247.
7. LoBue AD, Lindesmith L, Yount B, Harrington PR, Thompson JM, Johnston RE, Moe CL, Baric
RS: Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies
against multiple strains. Vaccine 2006;24:5220-5234.
8. Nicollier-Jamot B, Ogier A, Piroth L, Pothier P, Kohli E: Recombinant virus-like particles of a
norovirus (genogroup ii strain) administered intranasally and orally with mucosal adjuvants lt
and lt(r192g) in balb/c mice induce specific humoral and cellular th1/th2-like immune
responses. Vaccine 2004;22:1079-1086.