Molecular mechanisms of West Nile virus pathogenesis in brain cells.

We analyzed the response of human glioma cells to West Nile virus infection by investigating host transcriptional changes. Changes in expression of 23 genes showed similarities to those in other neurodegenerative diseases. These changes may be useful as potential biomarkers and elucidate novel mechanisms behind the neuropathology of infection with this virus.

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West Nile virus (WNV), a member of the family Flaviviridae, is the etiologic agent of West Nile fever. Since WNV is neurotropic, severe human meningoencephalitis is a common complication of infection and results in a considerable number of deaths. The medulla of the brainstem in the central nervous system (CNS) is the primary target of WNV (1).

WNV replicates in a wide variety of cell types, and studies have traditionally been carried out in Veto (green monkey kidney) and C6 (mosquito) cells. However, little work has been done with CNS cells. We conducted a global transcriptional analysis of human glioblastoma cell response to infection with WNV during peak virus production to determine the crucial virus-host interactions that take place during a severe neuroinvasive attack and identify putative mechanisms involved in WNV pathogenesis. The factors governing the development of neurologic disease, host immune response, patterns of clinical features, and outcomes are poorly understood in those infected with neurotropic flaviviruses (2).

A total of 173 genes were differentially expressed, many of which were not found in previous transcriptional studies of other flaviviruses (3). From these, 23 genes were identified that may play a role in cellular neurodegeneration. These novel changes induced by WNV may serve as biomarkers and help explain the neuropathologic features observed.

The Study

Most laboratory studies of WNV infections have been carried out in animal cell lines or human cell lines of non-CNS origins. In this study, human glioblastoma (A172) cells were found to be a useful laboratory model for investigating WNV infections. A172 (human glioblastoma) cells were maintained at 37[degrees]C in Dulbecco modified Eagle medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum. Confluent monolayers of A172 cells were infected with the Sarafend strain of WNV at a multiplicity of infection of 1. Twenty-four hours after infection, cells showed signs of cytopathic effects (cell-rounding) and produced high virus titer ([10.sup.8] PFU/mL). This demonstrated the highly susceptible nature of the neuroglial cells to WNV infection. Batches of cells were infected for microarray experiments, and a quantitative polymerase chain reaction was used to verify the reproducibility of the changes in gene expression.

Total cellular RNA was extracted from mock-infected and infected cells by using the RNeasy Mini kit and QIAshredder (Qiagen, Hilden, Germany). The CyScribe first-strand cDNA labeling kit (Amersham Biosciences, Piscataway, NJ, USA) was used to incorporate fluorescent Cy3-dCTP or Cy5-dCTP (Amersham Biosciences) into cDNA probes. The probes were subsequently purified by using CyScribe GFX purification columns (Amersham Biosciences). Equal amounts of labeled cDNA probes ([approximately equal to] 25 pmol) were combined for microarray hybridizations. Human 1A microarrays (Agilent Technologies, Palo Alto, CA, USA) were used, and hybridizations were performed on a Lucidea SlidePro Hybridizer (Amersham Biosciences). The microarray experiment was carried out in triplicate: 1 of the microarrays was with a dye-swap labeling to prevent skew in the results due to bias in CyDye incorporation.

Analyses of the scanned microarray images were performed with BRB ArrayTools version 3.1 (developed by R. Simon and A.P. Lam, National Cancer Institute, Bethesda, MD, USA, and available at http://linus.nci.nih.gov/BRB-ArrayTools.html), and normalized by using the Lowess method. A stringent lower limit threshold was set at 3 standard deviations of the pixel intensities of the negative control spots, and images were screened for changes in expression values of at least 2-fold. The differentially regulated genes were separately uploaded into EASE (4) to determine the biologic themes that were significantly over-represented (Fisher exact test with p values < 0.01). A total of 173 cellular genes were identified by ArrayTools to be differentially expressed in the WNV-infected A172 cells. EASE clustered 39 of the upregulated genes and 41 of the downregulated genes into specific functional groups (available at http://sps.nus.edu.sg/~kohweele/awn_genes.htm).

Functional classes that were found to be enriched in the upregulated genes encompassed those related to immunity, responses to external stimulus and pathogens, and apoptosis. Genes relating to the ubiquitin cycle, transcription regulation, and other physiologic processes were also identified by EASE. Functional classes that were downregulated were not commonly observed in a virus infection system. For instance, genes relating to the mitochondria, ribosomes, and protein biosynthesis were highly over-represented in down regulation (available at http://sps.nus. edu.sg/~kohweele/awn_genes.htm). From this set of genes, a group of 23 genes that may provide the molecular basis for the observed pathogenesis in the A172 cells was identified (Table 1).

A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to ensure an independent assessment of the microarray results. Genes for the qRT-PCR were selected to represent the broad spectrum of identified functional classes from the microarrays. The hypoxanthine guanine phosphoribosyltransferase gene was used as an internal control (primers for the PCR can be found at http://sps.nus.edu.sg/~kohweele/awn_genes.htm). RNA was reverse-transcribed by using SuperScript III (Invitrogen, Carlsbad, CA, USA), and a real-time PCR was carried out with Platinum SYBR Green (Invitrogen). A negative template control that contained all SYBR green reagents except DNA was performed in parallel on the iCycler iQ (Bio-Rad Laboratories, Hercules, CA, USA). The results corroborated the microarray data, thereby verifying the accuracy of the statistical analysis (Table 2). However, the qRT-PCR showed greater dynamism in fold changes than the microarray results because of the greater sensitivity of PCR compared with fluorescent detection.

Conclusions

In this study, WNV infection of human brain glioma cells showed advanced cytopathic effects within 24 h after infection and produced high virus yields. This demonstrated that human glioma cells from CNS are susceptible to WNV infection and are suitable for the study of viral pathogenesis.

The activation of the innate antiviral immune response pathways is often the primary cause of pathologic effects. The presence of double-stranded RNA replication complexes from viral origins causes the transcriptional activation of the interferon-[alpha]/[beta] (IFN-[alpha]/[beta]) or type-I IFN pathways (5). In this study on glioma cells, the activation of numerous interferon-induced proteins (such as IFIT1, IFIT2, IFI27, IFITM1, IFITM2, and G1P2) lends support to this mechanism of pathogenicity. Glial cells are useful in this study because they are immune cells of CNS origin. Activated glial cells have macrophagic activity and are primed to respond to the virus, therefore allowing the display of immune-mediated neuropathologic changes that reflect conditions in the natural CNS host cells. Glial cells can also activate the type-II (IFN-[gamma]) pathway and modulate the immune response by regulating cell trafficking of various leukocytes, including macrophage activation and stimulation of specific T cells responsible for cytotoxic immunity (6).

An example of this activation was finding that the HLA-C gene coding for the major histocompatibility complex class I (MHC-I) antigens was upregulated in the A 172 cells. Peptides derived from endogenous intracellular proteins are generally bound by the MHC-I molecules for presentation, thus paving the way for cell cytotoxicity in cellular immunity. In mice, the targeted killing of WNV-infected cells by CD8+ T cells may result in the severe neurologic disease often observed in WNV infections (7).

In addition, indoleamine 2,3 dioxygenase (INDO) was observed to be upregulated in WNV-infected A 172 cells. Increased production of INDO by glial cells causes neuronal injury in neuroinflammatory diseases (8). The upregulation of the pentaxin-related gene (PTX3) is also implicated in local tissue damage through the amplification of inflammation in innate immunity (9).

A group of genes causing apoptosis was also found to be upregulated, thus elucidating pathways linking virus replication to apoptosis. These genes include the tumor necrosis factor superfamily (TNFSF14), nuclear factor of [kappa] light-chain gene (NFKBIA), TNF receptor-associated factor (TRAF 1), and spermidine/spermine N1-acetyltransferase (SAT). This highly conserved process of cellular self-destruction serves to limit the spread of WNV (10).

A major group of genes relating to mitochondria was found to be downregulated. Mitochondrial defects due to respiratory-chain dysfunction and free-radical formation have been associated with neurodegenerative diseases such as Huntington disease, Parkinson disease, and Friedreich ataxia (11). Neurologic symptoms of these diseases were also observed in WNV-infected patients (12), suggesting similar neurodegenerative pathways.

The activity of genes belonging to the energy synthesis pathways was decreased. These genes included succinate dehydrogenase (SDHC), cytochrome c oxidase (COX5B/ COX6B), and various genes of the ATP synthase complex (ATP5G1, ATP5C1, ATP5J, ATP5B, ATP5A1, ATP50, and ATP5F1). Decreased energy production from the downregulation of these genes is known to cause severe neurodegeneration (13). Two antioxidant enzymes of the peroxiredoxin family (PRDX5 and PRDX3) were also downregulated. The increase in oxidative stress induced by reactive oxygen species can create a proinflammatory condition that results in CNS pathology and leads to Alzheimer disease and Down syndrome (14). Downregulation of the nascent polypeptide-associated complex (NACA) can also lead to similar neurodegeneration (15).

In summary, this global transcriptional study showed a complex network of WNV-induced A172 cell interactions during infection. The examination of glial A172 cell response has provided insights into the molecular mechanisms behind the observed neuronal pathology in WNV encephalitis.

Table 1. Differentially regulated genes involved in pathogenesis
of A172 cells infected with West Nile virus

                                                                  Fold
Gene                                   Gene name                 change

Immune response related
  OAS3                    2'-5'-oligoadenylate synthetase 3,       2.32
                                        100 kDa
  OASL                    2'-5'-oligoadenylate synthetase-like     3.46
  FIT1                      Interferon-induced protein with       10.74
                              tetratricopeptide repeats 1
  IFIT2                     Interferon-induced protein with        3.76
                              tetratricopeptide repeats 2
  IFI27                      Interferon, [alpha]-inducible         4.03
                                       protein 27
  IFITM1                   Interferon-induced transmembrane       12.00
                                    protein 1 (9-27)
  IFITM2                   Interferon-induced transmembrane        3.04
                                    protein 2 (1-8D)
  G1P2                       Interferon, [alpha]-inducible         9.50
                                protein (clone IFI-15K)
  HLA-C                    Major histocompatibility complex,       2.20
                                       class I, C
  INDO                    Indoleamine-pyrrole 2,3 dioxygenase      3.38
  PTX3                      Pentaxin-related gene, rapidly         3.44
                             induced by interleukin-1[beta]
Apoptosis related
  TNFSF14                 Tumor necrosis factor (TNF) (ligand)     2.19
                                 superfamily, member 14
  NFKBIA                     Nuclear factor of kappa light         4.13
                               polypeptide gene enhancer
  TRAF1                     TNF receptor-associated factor 1       2.01
  SAT                     Spermidine/spermine N1-acetyltrans-      2.18
                                         ferase
Mitochondria related
  SDHC                     Succinate dehydrogenase complex,       -2.31
                                       subunit C
  COX5B                     Cytochrome c oxidase subunit Vb       -2.13
  COX6B                     Cytochrome c oxidase subunit Vlb      -2.41
  ATP5G1                    ATP synthase, mitochondrial FO        -2.64
                             complex, subunit c, isoform 1
  ATP5C1                    ATP synthase, mitochondrial F1        -3.82
                             complex, [gamma] polypeptide 1
  ATP5J                     ATP synthase, mitochondrial FO        -2.11
                                  complex, subunit F6
  ATP5B                     ATP synthase, mitochondrial F1        -2.17
                              complex, [beta] polypeptide
  ATP5A1                    ATP synthase, mitochondrial F1        -2.21
                          complex, [alpha] subunit, isoform 1
  ATP5O                     ATP synthase, mitochondrial F1        -2.00
                                   complex, O subunit
  ATP5F1                    ATP synthase, mitochondrial FO        -2.43
                             complex, subunit b, isoform 1
  PRDX5                             Peroxiredoxin 5               -2.74
  PRDX3                             Peroxiredoxin 3               -2.27
Protein biosynthesis
    related
  NACA                      Nascent-polypeptide-associated        -2.17
                                  complex polypeptide

Table 2. Comparison of gene expression changes between microarray and
qRT-PCR in A172 cells infected with West Nile virus *

Gene                             Gene name

ARHI               DIRAS family, GTP-binding RAS-like 3
ATP5J       ATP synthase, mitochondrial FO complex, subunit F6
CEB1                       Hect domain and RLD 5
DNAJB1          DnaJ (Hsp40) homolog, subfamily B, member 1
DUSP1                 Dual specificity phosphatase 1
EGR1                      Early growth response 1
EIF4G2     Eukaryotic translation initiation factor 4 [gamma], 2
FLJ13855               Hypothetical protein FLJ13855
FOSL1                       FOS-like antigen 1
IFITM1       Interferon-induced transmembrane protein 1 (9-27)
LTA4H                    Leukotriene A4 hydrolase
RPL5                       Ribosomal protein L5
RPL7A                      Ribosomal protein L7a
RPLPO                  Ribosomal protein, large, PO
TFP12                Tissue factor pathway inhibitor 2

Gene       Microarray      RT-PCR
           fold change   fold change

ARHI          -2.72         -2.55
ATP5J         -2.11         -2.60
CEB1           2.32         42.22
DNAJB1        -1.97         -2.14
DUSP1          1.92          5.66
EGR1           4.79          8.57
EIF4G2        -2.11         -7.77
FLJ13855       2.05          3.85
FOSL1          2.08          6.50
IFITM1        12.03        527.61
LTA4H         -2.02         -8.10
RPL5          -2.97         -9.03
RPL7A         -2.03         -3.42
RPLPO         -2.15         -1.52
TFP12          5.21         11.58

* qRT-PCR, quantitative reverse transcription--polymerase
chain reaction.

Acknowledgments

We thank E.G. Westaway for providing the Sarafend strain of WNV.

This study was supported by the Biomedical Research Council (project no. 01/1/21/18/003).

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Wee-Lee Koh * and Mah-Lee Ng *

* National University of Singapore, Singapore

Mr. Koh is a postgraduate research student at the Department of Microbiology, National University of Singapore. His research interests include host-pathogen interactions and pathogenesis.

Dr. Ng is an associate professor at the Department of Microbiology, National University of Singapore. Her research interests include virology (mainly flaviviruses) and microscopic techniques.

Address for correspondence: Mah-Lee Ng, Flavivirology Laboratory, Department of Microbiology, National University of Singapore, 5 Science Dr 2, Singapore 117597; fax: [phone], email: [e-mail]