West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion

DENV infection does not affect tight junctions.

<p>A. MDCK cells were infected with DENV-2 and after 48 hours, samples were processed for indirect immunofluorescence using human anti-DENV serum and mouse anti-claudin-1. The human serum recognizes DENV structural proteins (DENV SP). Primary antibodies were detected using donkey anti-human Texas Red and donkey anti-mouse Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm. B. CACO2 cells were infected with DENV-2 and after 48 hours, relative levels of claudin-1, JAM-1, and occudin were determined by immunoblotting. DENV capsid protein was detected using a guinea pig polyclonal antibody and β-actin was detected using a mouse monoclonal antibody. Data from 3 independent experiments were averaged and plotted. Bars indicate standard error of the mean.</p

WNV infection leads to increased transcription of multiple tight junction genes.

<p>Total RNA extracted from mock and WNV-infected CACO-2 cells at 24, 48 and 72 h post-infection was subjected to quantitative RT-PCR. The levels of tight junction protein encoding mRNAs relative to GAPDH were determined using a comparative cT method. In the mock infected samples, values were normalized to 1.0 but are not shown on the graph. The bars and associated values denominate the mean values, with one-fold standard deviations depicted in the high and low bars.</p

Internalization of claudin-1 is blocked by disrupting microtubules or inhibiting dynamin function.

<p>MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using mouse anti-claudin-1 and rabbit anti-WNV capsid. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.</p

Expression of capsid in the absence of other WNV proteins does not cause degradation of tight junction proteins.

<p>CACO-2 (A) and MDCK (B) cells were infected with WNV or transduced with lentiviruses encoding GFP alone (Vector) or GFP and WNV capsid (Capsid). Forty eight hours post-infection/transduction, lysates were subjected to immunoblot analyses. Data from three independent experiments were used to determine the normalized level (to β-actin) of claudin-1, JAM-1, occludin in each sample. C. MDCK cells were infected with WNV and 48 hours later, lysates were subjected to immunoprecipitation (IP) with rabbit anti-capsid, anti-JAM-1 or mouse anti-claudin-1 antibodies followed by immunoblotting with antibodies to claudin-1, JAM-1 or capsid. WCL = whole cell lysate.</p

Internalization of JAM-1 is blocked by disrupting microtubules or inhibiting dynamin function.

<p>MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-JAM-1 and mouse anti-WNV NS3/2B. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.</p

Capsid expression does not result in redistribution of claudin-1 or JAM-1.

<p>MDCK cells were transduced with vector or capsid-expressing lentiviruses and at 48 hour post transduction, were processed for indirect immunofluorescence using mouse anti-claudin-1, mouse anti-occludin, rabbit anti-JAM-1, and rabbit or guinea pig anti-WNV capsid. Primary antibodies were detected using donkey anti-mouse Alexa546, donkey anti-rabbit Alexa647, and goat anti-guinea pig Alexa546 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bar = 10 µm.</p

List of Oligonucleotide Primers.

*<p>adjusted for 50 mM salt concentration.</p

WNV-induced degradation of claudin-1 and JAM-1 requires dynamin and microtubules.

<p>A. CACO-2 cells were infected with WNV and 24 hours later were treated with nocodazole (10 µM), Dynasore (10 µM), paclitaxel (1 µM), latrunculin B (10 µM) or DMSO for a further 8 hours. The corresponding cell lysates then subjected to immunoblot analyses. B. Data from three independent experiments were used to determine the normalized levels of claudin-1 and JAM-1 (relative to β-actin).</p