Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export

Virology Journal

Laura L Newcomb¹  Hilario Medina-Ramirez¹  Adeba Mohammad¹  Veronica Perez¹  Steven Bui¹  Sean Larsen¹ 

[1] Department of Biology, California State University San Bernardino, 5500 University Parkway, San Bernardino, CA 92407, USA

关键词: Nuclear export;    mRNA;    Virus;    Influenza;   

Others  :  1148525
DOI  :  10.1186/1743-422X-11-154

 received in 2014-02-25, accepted in 2014-08-12,  发布年份 2014

【 摘 要 】

Background

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1.

Methods

Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription - quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA.

Results

Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export.

Conclusions

These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.

【 授权许可】

   
2014 Larsen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Centers for Disease Control [http://www.cdc.gov/flu/ webcite]
• [2]Lagacé-Wiens PRS, Rubinstein E, Gumel A: Influenza epidemiology – past, present, and future. Crit Care Med 2010, 38(4):e1-e9. PMID: 20029350
• [3]Russell CA, Fonville JM, Brown AE, Burke DF, Smith DL, James SL, Herfst S, van Boheemen S, Linster M, Schrauwen EJ, Katzelnick L, Mosterín A, Kuiken T, Maher E, Neumann G, Osterhaus AD, Kawaoka Y, Fouchier RA, Smith DJ: The potential for respiratory droplet-transmissible A/H5N1 influenza virus to evolve in a mammalian host. Science 2012, 336(6088):1541-1547. PMID: 22723414
• [4]Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA: Airborne transmission of influenza A/H5N1 virus between ferrets. Science 2012, 336(6088):1534-1541. PMID: 22723413
• [5]Plotch SJ, Bouloy M, Ulmanen I, Krug RM: A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 1981, 23(3):847-858. PMID: 6261960
• [6]Poon LL, Pritlove DC, Fodor E, Brownlee GG: Direct evidence that the poly (A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J Virol 1999, 73(4):3473-3476. PMID: 10074205
• [7]Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR: The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 1989, 338(6212):254-257. PMID: 2784194
• [8]Fornerod M, Ohno M, Yoshida M, Mattaj IW: CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 1997, 90(6):1051-1060. PMID: 9323133
• [9]Gruter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi A, Wilm M, Felber BK, Izaurralde E: TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell 1998, 1(5):649-659. PMID: 9660949
• [10]Pasquinelli AE, Ernst RK, Lund E, Grimm C, Zapp ML, Rekosh D, Hammarskjold ML, Dahlberg JE: The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J 1997, 16(24):7500-7510. PMID: 9405378
• [11]Bogerd HP, Echarri A, Ross TM, Cullen BR: Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J Virol 1998, 72(11):8627-8635. PMID: 9765402
• [12]Hao L, Sakurai A, Watanabe T, Sorensen E, Nidom CA, Newton MA, Ahlquist P, Kawaoka Y: Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 2008, 454(7206):890-893. PMID: 18615016
• [13]Read EK, Digard P: Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export. J Gen Virol 2010, 91(5):1290-1301. PMID: 20071484
• [14]Wang W, Cui ZQ, Han H, Zhang ZP, Wei HP, Zhou YF, Chen Z, Zhang XE: Imaging and characterizing influenza A virus mRNA transport in living cells. Nucleic Acids Res 2008, 36(15):4913-4928. PMID: 18653528
• [15]Satterly N, Tsai PL, van Deursen J, Nussenzveig DR, Wang Y, Faria PA, Levay A, Levy DE, Fontoura BM: Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc Natl Acad Sci U S A 2007, 104(6):1853-1858. PMID: 17267598
• [16]Neumann G, Hughes MT, Kawaoka Y: Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J 2000, 19(24):6751-6758. PMID: 11118210
• [17]Elton D, Simpson-Holley M, Archer K, Medcalf L, Hallam R, McCauley J, Digard P: Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway. J Virol 2001, 75(1):408-419. PMID: 11119609
• [18]Amorim MJ1, Read EK, Dalton RM, Medcalf L, Digard P: Nuclear export of influenza A virus mRNAs requires ongoing RNA polymerase II activity. Traffic 2007, 8(1):1-11. PMID: 17132145
• [19]Kang Y, Cullen BR: The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. Genes Dev 1999, 13(9):1126-1139. PMID: 10323864
• [20]Huang Y, Steitz JA: Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol Cell 2001, 7(4):899-905. PMID: 11336712
• [21]Min JY, Li S, Sen GC, Krug RM: A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 2007, 363(1):236-243. PMID: 17320139
• [22]Robb NC, Fodor E: The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein. J Gen Virol 2012, 93(Pt 1):113-118. PMID: 21918006
• [23]Schneider J, Wolff T: Nuclear functions of the influenza A and B viruses NS1 proteins: do they play a role in viral mRNA export? Vaccine 2009, 27(45):6312-6316. PMID: 19840666
• [24]de la Luna S, Fortes P, Beloso A, Ortín J: Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J Virol 1995, 69(4):2427-2433. PMID: 7884890
• [25]García-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE, Palese P, Muster T: Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 1998, 252(2):324-330. PMID: 9878611
• [26]Nemeroff ME, Barabino SM, Li Y, Keller W, Krug RM: Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3’end formation of cellular pre-mRNAs. Mol Cell 1998, 1(7):991-1000. PMID: 9651582
• [27]Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, Hartmann E, Görlich D: Identification of a tRNA-specific nuclear export receptor. Mol Cell 1998, 1(3):359-369. PMID: 9660920
• [28]Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U: Nuclear export of microRNA precursors. Science 2004, 303(5654):95-98. PMID: 14631048
• [29]Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez DR, Donis R, Hoffmann E, Hobom G, Kawaoka Y: Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A 1999, 96(16):9345-9350. PMID: 10430945