Imaging and characterizing influenza A virus mRNA transport

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http://nar.oxfordjournals.org/cgi/content/full/36/15/4913


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Imaging and characterizing influenza A virus mRNA transport in living cells

<nobr>Wei Wang¹^,2</nobr>, <nobr>Zong-Qiang Cui¹</nobr>, <nobr>Han Han¹^,2</nobr>, <nobr>Zhi-Ping Zhang¹</nobr>, <nobr>Hong-Ping Wei¹</nobr>, <nobr>Ya-Feng Zhou¹</nobr>, <nobr>Ze Chen¹</nobr> and <nobr>Xian-En Zhang¹^,*</nobr> ¹State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 and ²Graduate School, Chinese Academy of Sciences, Beijing 100039, China
[SIZE=-1]*To whom correspondence should be addressed. Tel: +86 10 5888 1508; Fax: +86 10 5888 1559; Email: x.zhang@wh.iov.cn<script type="text/javascript"><!-- var u = "x.zhang", d = "wh.iov.cn"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>[/SIZE]
[SIZE=-1]Received April 4, 2008.[/SIZE] [SIZE=-1]Revised June 23, 2008.[/SIZE] [SIZE=-1]Accepted July 8, 2008.[/SIZE]

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</td> <th valign="middle" width="95%" align="left">[SIZE=+2] ABSTRACT [/SIZE]</th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left">[SIZE=-1]
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The mechanisms of influenza A virus mRNA intracellular transportare still not clearly understood. Here, we visualized the distributionand transport of influenza A virus mRNA in living cells usingmolecular beacon (MB) technology. Confocal-FRAP measurementsdetermined that the transport of influenza A virus intronlessmRNA, in both nucleus and cytoplasm, was energy dependent, beingsimilar to that of Poly(A)⁺ RNA. Drug inhibition studies inliving cells revealed that the export of influenza A virus mRNAis independent of the CRM1 pathway, while the function of RNApolymerase II (RNAP-II) may be needed. In addition, viral NS1protein and cellular TAP protein were found associated withinfluenza A virus mRNA in the cell nucleus. These findings characterizeinfluenza A virus mRNA transport in living cells and suggestthat influenza A virus mRNA may be exported from the nucleusby the cellular TAP/p15 pathway with NS1 protein and RNAP-IIparticipation.
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</td> <th valign="middle" width="95%" align="left">[SIZE=+2] INTRODUCTION [/SIZE]</th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left">[SIZE=-1]
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Influenza virus is one of the few RNA viruses to synthesizeits mRNA in the nucleus of infected cells (1). The virus mRNAsare potential substrates for the cellular splicing machineryand need to be exported from the nucleus to enable the viralproteins to be synthesized (2). Uncovering the mechanisms ofinfluenza virus mRNA export is of great importance to trulyunderstand the replication and pathogenicity of the virus. Thenuclear export of cellular mRNA is mediated by several proteinsthat bind to mRNA and to pre-mRNA precursors (3). However, unlikecellular intron-containing mRNAs, most influenza virus mRNAsare intronless. Hence, the export mechanisms of viral intronlessmRNAs might be different from those of cellulr mRNAs. Moreover,because there are three different types of influenza virus mRNA,more than one mechanism of nuclear export might operate in virus-infectedcells (1). The first type of influenza virus mRNA includes intronlessmRNAs, such as PA, PB1, PB2, HA, NA and NP mRNA. The secondtype of viral mRNA consists of the M1 and NS1 mRNAs, which containintrons but do not undergo splicing. The M2 and NS2 mRNAs, whichare produced by splicing, comprise the third type of viral mRNA.The mechanisms of the nuclear export of these three types ofinfluenza A virus mRNA remain unknown.
Two pathways have been described that appear to be responsiblefor the export of viral mRNA (4). The first RNA export pathwaywas the CRM1 pathway, which is utilized by human immunodeficiencyvirus (HIV) through the mediation of the Rev protein (5). Herpessimplex virus (HSV) also utilizes CRM1 to export its mRNA (6).However, other studies showed that CRM1 may be not a major contributorto mRNA export in metazoans or yeast (7,8). The human proteinTAP, and its yeast ortholog Mex67p, might be the best candidatesfor mRNA export receptors because they shuttle between the nucleusand cytoplasm, cross-link to poly(A)⁺ RNA, localize at the nuclearpores and interact directly with nucleoporins (9?12).The TAP pathway was reported to be used by HSV ICP27 to exportits intronless mRNAs (4). Moreover, TAP protein could also promotethe export of constitutive transport element (CTE) containingtranscripts of some virus such as type D retrovirus (9,10,13).Influenza A virus mRNA may, therefore, be exported from thenucleus by the CRM1 dependent pathway or by the TAP/p15 pathway.Previous studies have shown that influenza virus NS1 proteincould selectively inhibit cellular mRNA export by binding withCPSF and PABII (1), or by forming an inhibitory complex withcellular mRNA export factors TAP and p15 (14). Moreover, NS1can also inhibit the splicing and export of its own mRNA, inan RNA binding-dependent manner (2). Nevertheless, the mechanismsby which influenza virus mRNAs are exported from the nucleusand the roles of viral NS1 protein in influenza A virus intronlessmRNA export are still unclear.
The ability to accurately and repeatedly track mRNA in livingmammalian cells would help us to fully understand the mRNA transportmechanism. There are a variety of tools currently used to visualizeintracellular mRNAs, including molecular beacons (MBs) and fluorescentlylabeled oligonucleotide probes. MBs are a powerful and simpletool for cellular mRNA and viral RNA visualization in livingcells (15?21). Live-cell imaging of mRNA could shed lighton many fundamental processes, such as the kinetics of mRNAproduction, mRNA localization and transportation inside a celland cellular responses to virus infection and to virus?hostinteraction. We, therefore, used MBs as a detection probe totrack influenza A virus mRNA in living host cells, in orderto explore the mechanisms of viral mRNA export.
In this study, we successfully visualized influenza A virusmRNA in living mammalian cells, and studied the dynamic behaviorsof influenza virus mRNA by Confocal-FRAP experiments. By imagingexperiments of living cells and protein immunofluorescence analysisin fixed cells, it was found that influenza A virus mRNAs couldcolocalize with viral NS1 and cellular TAP protein in cell nucleus.Moreover, coimmunoprecipitation experiments of influenza A virusmRNAs with NS1 and TAP protein revealed that NS1 and TAP proteincould be physically associated with both intron-containing andintronless mRNAs of influenza A virus. By performing ActinomycinD (ActD) inhibition experiments in living cells we observedthat RNA polymerase II (RNAP-II) and other factors might beinvolved in influenza virus mRNA export. Furthermore, LeptomycinB (LMB), a specific inhibitor of CRM1, could not inhibit influenzaA virus mRNA export in living cells. Therefore, influenza Avirus mRNA export may be independent of CRM1. Together, theseresults indicate that the cellular transport of influenza Avirus mRNA may be energy dependent and utilize the cellularTAP/p15 transport pathway with the participation of viral NS1protein and cellular RNAP-II.