Andes virus regulation of cellular microRNAs contributes to hantavirus-induced endothelial cell permeability

Hantaviruses infect human endothelial cells (ECs) and cause two diseases marked by vascular permeability defects, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Vascular permeability occurs in the absence of EC lysis, suggesting that hantaviruses alter normal EC fluid barrier functions. ECs infected by pathogenic hantaviruses are hyperresponsive to vascular endothelial growth factor (VEGF), and this alters the fluid barrier function of EC adherens junctions, resulting in enhanced paracellular permeability. Vascular permeability and VEGF-directed responses are determined by EC-specific microRNAs (miRNAs), which regulate cellular mRNA transcriptional responses. miRNAs mature within cytoplasmic processing bodies (P bodies), and the hantavirus nucleocapsid (N) protein binds RNA and localizes to P bodies, suggesting that hantaviruses may modify miRNA functions within infected ECs. Here we assessed changes in EC miRNAs following infection by the HPS-causing Andes hantavirus (ANDV). We analyzed 352 human miRNAs within ANDV-infected ECs using quantitative real-time (RT)-PCR arrays. Fourteen miRNAs, including six miRNAs that are associated with regulating vascular integrity, were upregulated >4-fold following infection by ANDV. Nine miRNAs were downregulated 3- to 3,400-fold following ANDV infection; these included miR-410, involved in regulating secretion, and miR-218, which is linked to the regulation of EC migration and vascular permeability. We further analyzed changes in miR-126, an EC-specific miRNA that regulates vascular integrity by suppressing SPRED1 and PIK3R2 mRNAs. While miR-126 levels were only slightly altered, we found that SPRED1 and PIK3R2 mRNA levels were increased 10- and 7-fold, respectively, in ANDV-infected ECs but were unaltered in ECs infected by the nonpathogenic Tula hantavirus (TULV). Consistent with increased SPRED1 expression, we found that the level of phospho-cofilin was decreased within ANDV-infected ECs. Moreover, small interfering RNA (siRNA) knockdown of SPRED1 dramatically decreased the permeability of ANDV-infected ECs in response to VEGF, suggesting that increased SPRED1 contributes to EC permeability following ANDV infection. These findings suggest that interference with normal miRNA functions contributes to the enhanced paracellular permeability of ANDV-infected ECs and that hantavirus regulation of miRNA functions is an additional determinant of hantavirus pathogenesis.