LETTERS. Interferon modulation of cellular micrornas as an antiviral mechanism

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1 Vol Octoer 27 doi:1.138/nture625 LETTERS Interferon modultion of cellulr micrornas s n ntivirl mechnism Irene M. Pedersen 1, Guofeng heng 3, Stefn Wielnd 3, Stefno Volini 4, rlo M. roce 4, Frncis V. hisri 3 & Michel Dvid 1,2 RNA interference through non-coding micrornas (mirnas) represents vitl component of the innte ntivirl immune response in plnts nd inverterte nimls; however, role for cellulr mirnas in the defence ginst virl infection in mmmlin orgnisms hs thus fr remined elusive 1. Here we show tht interferon et (IFN) rpidly modultes the expression of numerous cellulr mirnas, nd tht eight of these IFN-induced mirnas hve sequence-predicted trgets within the heptitis virus (HV) genomic RNA. The introduction of synthetic mirna-mimics corresponding to these IFN-induced mirnas reproduces the ntivirl effects of IFN on HV repliction nd infection, wheres neutrliztion of these ntivirl mirnas with nti-mirnas reduces the ntivirl effects of IFN ginst HV. In ddition, we demonstrte tht IFN tretment leds to significnt reduction in the expression of the liver-specific 22, n mirna tht hs een previously shown to e essentil for HV repliction 2. Therefore, our findings strongly support the notion tht mmmlin orgnisms too, through the interferon system, use cellulr mirnas to comt virl infections. mirnas re clss of smll non-coding RNA molecules tht function through post-trnscriptionl regultion of gene expression y process termed RNA interference (RNAi). Over 5 mirnaencoding genes, which seem to e exclusively trnscried y RNA polymerse II, hve een identified in mmmls. These primry micrornas re processed y the enzymes Drosh/DGR8/ RNASEN into hirpin-loop-contining pre-mirnas, which re then suject to nucler export vi exportin 5. Further enzymtic processing of the pre-mirnas y Dicer leds to mture mirna duplex tht is loded into the RNA-induced silencing complex (RIS), in which the mirna guides RIS to complementry messenger RNAs. On the sis of the degree of homology etween the mirna nd the mrna, RIS cn inhiit mrna function y either promoting its clevge or y inhiiting its trnsltion 3,4. Prticulrly, the sequence complementrity in the 6 8 se pir seed region t the 59 end of the mirna mrna heteroduplex seems to determine the specificity of mirna trgetrna interctions 5. RNAi-medited trgeting of virl RNAs ws first recognized in plnts s n ntivirl defence mechnism, ut it is now pprent tht invertertes lso use RNAi to comt virl infection 1,6,7. However, thus fr, no evidence hs een presented in support of protective RNAi-sed ntivirl response in mmmlin cells. Rther, the hypothesis hs een proposed tht the extremely potent interferon system hs displced RNAi s the key defence ginst virus infection in mmmlin cells 1. Indeed, severl type I interferon (IFN/)- regulted gene products such s protein kinse R, the 2-5 OA synthse/rnse L system, the denosine deminse ADAR1 or the Mx GTPses re importnt contriutors to the ntivirl properties of these cytokines 8,9. However, the possiility tht IFN/ might induce cellulr mirnas tht trget virl trnscripts nd therey use RNAi s prt of their rsenl ginst invding viruses hs een left unexplored. To test whether IFN/ could lter the expression of cellulr mirnas, we used microrry technology to nlyse RNA derived from IFN/- or IFNc-stimulted cells. This initil screening effort identified,3 mirnas, the expression levels of which were incresed or ttenuted in response to IFN/ or IFNc. Becuse we were interested in determining whether these mirnas could potentilly contriute to the ntivirl effects of interferons, we performed sequence complementrity nlysis of these mirnas ginst virl trnscripts or virl genomic RNAs with n initil focus on the crucil seed sequence. This pproch reveled promising mtches mong severl viruses, most of which hrour n RNA-sed genome. Specificlly, eight of the IFN-induced mirnas (, mir-3, 28,,,, mir-431 nd ) displyed nerly perfect complementrity in their seed sequences with heptitis virus (HV) RNA genomes. This finding ws rther intriguing considering tht IFN nd IFN re the most common tretment regimen for HV infection 1,11. Interestingly, similr nlysis of the heptitis B virus ( DNA virus) yielded no significnt mtches. HV is the sole memer of the hepcivirus genus of the Flviviridie fmily, nd is represented y six mjor genotypes. The virion contins 9.6 k single-strnded RNA genome of positive polrity, with highly invrint 59 nd 39 untrnslted regions 12,13. After virus entry into the host cell, the virl genome is uncoted nd serves s templte for the trnsltion of single polyprotein, which is susequently processed y host nd virl proteses. The nonstructurl virl proteins then initite the synthesis of negtivestrnd RNA, which serves s repliction templte for the genertion of new positive-strnd virl genomes 12,13. Sequence lignment of the six HV genotypes illustrted tht the puttive mirna trget sites for the IFN-induced mirnas re locted in res strictly conserved mong ll HV genotypes (see exmples in Supplementry Fig. 1) s well s in regions tht differ etween the genotypes such tht high seed-sequence complementrity occurs only with selected HV genotypes (see exmples in Supplementry Fig. 1). To verify our microrry studies we nlysed mirna induction in response to IFN in the humn heptom cell line Huh7, s well s in freshly isolted primry murine heptocytes y quntittive PR (qpr). As shown in Fig. 1,, IFN tretment resulted in similr induction of the prospective ntivirl mirnas in oth cell types to tht oserved in, well-chrcterized IFN/-regulted gene 14. Two mirnas (25 nd 42) tht were found 1 Deprtment of Moleculr Biology, Division of Biologicl Sciences, nd 2 Moores ncer enter, University of liforni Sn Diego, L Joll, liforni 9293, USA. 3 Division of Experimentl Pthology, The Scripps Reserch Institute, L Joll, liforni 9237, USA. 4 Deprtment of Moleculr Virology, Immunology & Medicl Genetics, Ohio Stte University, olumus, Ohio 4321, USA. 27 Nture Pulishing Group 919

2 LETTERS NATURE Vol Octoer 27 c d Per cent of control ,2 1, , e Huh7 22 mir ontrol Per cent of control Primry heptoctyes mir ,8 5 1,5 4 1, ontrol ontrol ontrol ontrol ontrol , ontrol , ontrol , ontrol , ontrol Figure 1 Regultion of mirna expression y IFN in Huh7 cells nd primry heptocytes.,, Huh7 cells () or primry heptocytes () were stimulted with 1 U ml 21 IFN for 2 h, nd the indicted mirnas were quntified y qpr. induction is shown for comprison. c, Time course of mirna induction y IFN: Huh7 cells were stimulted with 1 U ml 21 IFN for the indicted times, nd, or expression ws quntified y qpr. d, Dose response nlysis of mirna induction y IFN: Huh7 cells were stimulted with the indicted doses of IFN for 2 h, nd, or expression ws quntified y qpr. e, Time course nd dose response nlysis of 22 downregultion y IFN: Huh7 cells were stimulted s descried in c nd d, nd 22 ws quntified y qpr. Error rs, mens 6 s.d. of t lest four independent experiments. IFN-unresponsive in the microrry nlysis were included s negtive controls. We next performed kinetic, s well s dose response, nlyses of the induction of mirnas y IFN. Time course nlysis reveled tht induction of nd occurs very rpidly, with pek concentrtions within 3 min, nd thus even precedes the upregultion of (Fig. 1c). In similr mnner to induction, upregultion of nd followed clssicl dose response curve etween 1 nd 1, U ml 21 IFN (Fig. 1d), nd could e locked y ctinomycin D (dt not shown), indicting tht the incresed levels of mirnas re the result of trnscriptionl induction. 22 is specificlly expressed in the liver, nd previous studies using nti-mirnas elegntly demonstrted tht 22, nd consequently Dicer 15, re essentil for HV repliction 2. We therefore tested whether 22 ws lso suject to regultion y IFN. As shown in Fig. 1e, IFN-stimultion of Huh7 cells resulted in trnsient, ut pronounced (,8%) down-modultion of 22. In similr mnner to mirna induction, 1 U ml 21 IFN induced mximl ttenution of 22 expression, nd no dditionl effect ws oserved with incresed IFN concentrtions (Fig. 1e). To evlute whether the eight IFN-induced mirnas with sequence mtches in the HV genome re indeed cple of inhiiting HV repliction, we trnsfected synthetic mirna-mimics corresponding to these mirnas into Huh-7 cells tht hrour n utonomously replicting, dicistronic full-length HV replicon 16,17. An nti-mirna ginst 22 served s positive control, ecuse it hd previously een shown to significntly reduce the undnce of replicon RNA in this system 2. As expected, non-specific control mirnas (individully or comined) or nti-mirna oligos did not lter the mounts of HV replicon RNA, wheres introduction of nti-22 resulted in,7% reduction in virl RNA levels, s previously reported (Fig. 2). Trnsfection of the eight cndidte mirnas individully reveled tht mirnas,,, mir-431 nd were indeed le to sustntilly 92 (mix5) Anti- mir-3 28 mir-431 Anti-22 mir-mix5 mir-mix5 + nti-22 Anti-22 mir-mix5 mir-mix5 + nti Nture Pulishing Group Figure 2 IFN-induced mirnas disply nti-virl ctivity ginst HV., JFH1-replicon-contining Huh7 cells were trnsfected with single nonspecific mirna-mimics (), pool of control mirnas (mirctrl(mix5)) or nti-mirnas (nti-), or specific mirna-mimics corresponding to the eight IFN-induced mirnas, or specific nti-mirnas. In ddition, comintion of the five mirnas tht displyed nti-virl ctivity individully ws used (mir-mix5) with or without nti-22, s indicted, nd HV RNA ws quntified y qpr fter 48 h.,smes, except Huh7 cells were infected with live JFH-1 virus for 48 h (error rs, mens 6 s.d. of t lest four independent experiments; P vlues re from pired Student s t-tests). P =.1 P <.1

3 NATURE Vol Octoer 27 LETTERS ttenute virl repliction, wheres mirnas, mir-3 nd 28 were without effect (Fig. 2). Trnsfection of mixture of the five functionl mirna-mimics yielded.8% reduction in virl RNA lod. As IFN induces mirnas,,, mir-431 nd ut downregultes 22, we decided to imitte this cellulr response y trnsfecting the mix of mirnamimics in comintion with nti-22. Indeed, smll, ut reproducile dditive inhiitory effect on HV repliction ws oserved tht ws similr in efficcy to tretment of the cells with IFN (Fig. 2). Virtully identicl results were otined when Huh7 cells were infected with live JFH1 HV nd either virl RNA levels (Fig. 2) or virl foci formtion (Supplementry Fig. 2) were nlysed, further corroorting the fct tht the IFN-induced ltertions in the mirna expression profile endow the cells with pronounced ntivirl stte. To investigte whether these nti-virl mirnas re trgeting the HV genome rther thn inducing non-specific ntivirl stte through ltertion of cellulr gene expression, we took dvntge of the fct tht the J6F HV moleculr clone hs single-nucleotide vritions in the predicted trget seed sequences for nd s opposed to JFH1 (Fig. 3). As J6F is non-infectious in vitro, we employed n infection-competent J6F/JFH1 chimeric virus tht crried the mutnt nd trget sites (chimer J6/JFH) (Fig. 3). As nticipted, ws effective ginst JFH1, ut not ginst J6/JFH, which contined the mutnt trget site. Similrly, only inhiited the repliction of JFH1 contining the correct trget site, ut ws ineffective ginst J6/JFH (Fig. 3). Introduction of compenstory single nucleotide chnge into nd (designted * nd *) to mtch their seed sequence to J6/JFH yielded reversed efficcy profile compred to the wild-type mirnas when they were tested ginst the two viruses. As such, oth * nd * were unle to sudue repliction of JFH1, ut clerly inhiited repliction of J6/JFH (Fig. 3). Together, these results suggest tht t lest nd re directly trgeting the HV genomic RNA. Although the ove descried experiments demonstrte tht IFN-induced mirnas, in conjunction with the downregultion of 22, re sufficient to induce n ntivirl stte, the question remined whether this modultion of cellulr mirna levels ws necessry for IFN to prevent HV repliction. To this end, we decided to counterct the IFN-elicited chnges in mirna expression y trnsfecting nti-mirnas ginst mirnas,,, mir-431 nd, with nd without the inclusion of n 22 mimic. IFN tretment leds to.9% reduction in the mount of virl HV replicon RNA nd this inhiition is unffected y trnsfected non-specific control nti-mirnas. As shown in Fig. 4, introduction of the nti-mirna mix, or of the 22 mimic seprtely, ttenuted the IFN effect to,75% inhiition. o-trnsfection of the nti-mirna mix nd the 22 mimic further reduced the efficcy of IFN to,5%, indicting tht mir-trl * * * * * (81) JFH1: AA U A GUU J6F: AA A A GUU JFH1 J6/JFH J6F (6,68) JFH1: UUGAUGG A A J6F: UUGAUGG G A Figure 3 IFN-induced mirnas directly trget virl genomic RNA.,, An infectious chimeric virus ws constructed from JFH1 nd J6F s shown in ; the numers in rckets indicte the loction in the virion (nucleotides) tht the mirna inds. The Huh7-cell suclone Huh7.5.1c2 ws trnsfected with either or, or with the mutnt * nd * hrouring compenstory muttion in the seed sequence s outlined in. Trnsfected Huh7.5.1c2 cells were infected with JFH1(D183) (ref. 23) or chimeric J6/JFH, nd HV RNA ws quntified y qpr fter 24 or 6 h post infection, respectively, during the phse of exponentil virl RNA mplifiction, to ccommodte the difference in repliction kinetics etween the two viruses (error rs, mens 6 s.e.m. of 8 independent experiments; P vlues re from pired Student s t-tests). * Anti-22 JFH1 J6/JFH JFH1 * * J6/JFH * * trl-mir trl-mir P vlues (pired t-test) 27 Nture Pulishing Group 921

4 LETTERS NATURE Vol Octoer 27 Anti-(5) Anti-miR-mix5 22 Anti-miR-mix Anti- + Anti-miR-mix Anti-miR-mix Figure 4 IFN-induced mirnas medite nti-virl IFN responses ginst HV. JFH1-replicon-contining Huh7 cells were trnsfected with nonspecific mirna-mimics (), or non-specific nti-mirnas (nti-mirctrl), or pool of nti-mirnas (nti-(5)), or comintion of ntimirna complementry to the five IFN-induced mirnas nd with potent nti-virl effect (nti-mirna-mix5), nd/or with specific mirnas nd ntimirnas, s indicted, efore stimultion with IFN for 48 h. HV RNA ws quntified y qpr (mens 6 s.d. of t lest four independent experiments; P vlues re from pired Student s t-tests). modultion of the expression levels of the identified mirnas hs n importnt, leit not exclusive, role in the ntivirl effects of IFN ginst HV. It remins to e determined whether ltertions in the expression levels of the identified cellulr mirnas ccount for only prt of the ntivirl response, or if dditionl unidentified mirnas tht hve not een neutrlized y nti-mirnas medite the remining ntivirl IFN effects. In summry, our results demonstrte tht IFN/ upregultes severl cellulr mirnas tht re cple of inhiiting HV repliction nd infection. In ddition, downregultion of 22 in response to IFN further contriutes to the ntivirl effects of this cytokine. These findings not only offer new model of the host defence mechnisms tht exist in mmmlin cells, ut lso dd new component to the ntivirl rsenl employed y interferons. Furthermore, lthough mple documenttion is present in the literture for the developmentlly regulted or tissue-specific expression of mirnas nd their dysregultion in mlignnt cells 18,19, little evidence existed for direct nd immedite trnscriptionl regultion of mirnas y n endogenous lignd such s cytokine or growth fctor. Our results provide some of the first evidence for rpid chnges in cellulr mirna levels in response to lignd stimultion. During the preprtion of this mnuscript, it ws reported tht IFN induced upregultion of 55 (ref. 2), mirna with, s of yet, no identified trgets. The delyed induction kinetics oserved in their studies supports the model tht 55 upregultion is medited y TNF s prt of n utocrine response elicited y IFN, rther thn y IFN itself 2. Nevertheless, this study nd our present report provide the first exmples of rpid modultion of cellulr mirnas s components of the mmmlin innte immune response. METHODS SUMMARY ell culture. The humn heptom-derived cell lines Huh7 nd their JFH-1- replicon-contining suclone were mintined in DMEM with 1% FS/1mM Hepes, penicillin/streptomycin nd 2 mm L-glutmine. The JFH-1 full-length genomic replicon construct pfgr-jfh-1 ws otined from T. Wkit 21.A Huh7 cell clone tht stly replictes the full-length genomic HV RNA ws selected nd used in these experiments, s descried previously 22. The Huh7.5.1c2 cell line ws derived from curing replicon-contining Huh cells y interferon tretment. Primry murine heptocytes (Bl) were collected fter collgense perfusion of the livers for 3 min t 37 u. mirna rry nlysis. Microrry nlysis of totl RNA of interferonstimulted lymphocytes ws performed t the Ohio Stte University omprehensive ncer enter Microrry Shred Resource, s descried 23. P =.1 RNA extrction. Totl RNA ws isolted using TriZol ccording to the mnufcturer s instructions. Rel-time PR. Quntifiction of HV genomic RNA ws performed using HV- nd -ctin- or GAPDH-specific primers s descried 16. Rel-time PR-sed quntifiction of mirnas ws performed using mirna nlysis kits specific for ech individul mirna (Applied Biosystems), ccording to the mnufcturers instructions. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 26 July; ccepted 29 August ullen, B. R. Is RNA interference involved in intrinsic ntivirl immunity in mmmls? Nture Immunol. 7, (26). 2. Jopling,. L., Yi, M., Lncster, A. M., Lemon, S. M. & Srnow, P. Modultion of heptitis virus RNA undnce y liver-specific microrna. Science 39, (25). 3. Brtel, D. P. MicroRNAs: genomics, iogenesis, mechnism, nd function. ell 116, (24). 4. Amros, V. The functions of niml micrornas. Nture 431, (24). 5. Lewis, B. P., Burge,. B. & Brtel, D. P. onserved seed piring, often flnked y denosines, indictes tht thousnds of humn genes re microrna trgets. ell 12, 15 2 (25). 6. Zmore, P. D. Plnt RNAi: How virl silencing suppressor inctivtes sirna. urr. Biol. 14, R198 R2 (24). 7. Bulcome, D. RNA silencing in plnts. Nture 431, (24). 8. Ktze, M. G., He, Y. & Gle, M. Jr. Viruses nd interferon: fight for supremcy. Nture Rev. Immunol. 2, (22). 9. Smuel,. E. Antivirl ctions of interferons. lin. Microiol. Rev. 14, (21). 1. Ling, T. J., Rehermnn, B., Seeff, L. B. & Hoofngle, J. H. Pthogenesis, nturl history, tretment, nd prevention of heptitis. Ann. Intern. Med. 132, (2). 11. Luer, G. M. & Wlker, B. D. Heptitis virus infection. N. Engl. J. Med. 345, (21). 12. Wielnd, S. F. & hisri, F. V. Stelth nd cunning: heptitis B nd heptitis viruses. J. Virol. 79, (25). 13. Rehermnn, B. & Nscimeni, M. Immunology of heptitis B virus nd heptitis virus infection. Nture Rev. Immunol. 5, (25). 14. Lrner, A.. et l. Trnscriptionl induction of two genes in humn cells y et interferon. Proc. Ntl Acd. Sci. USA 81, (1984). 15. Rndll, G. et l. ellulr cofctors ffecting heptitis virus infection nd repliction. Proc. Ntl Acd. Sci. USA 14, (27). 16. Zhong, J. et l. Roust heptitis virus infection in vitro. Proc. Ntl Acd. Sci. USA 12, (25). 17. Mordpour, D. et l. Insertion of green fluorescent protein into nonstructurl protein 5A llows direct visuliztion of functionl heptitis virus repliction complexes. J. Virol. 78, (24). 18. lin, G. A. & roce,. M. MicroRNA cncer connection: the eginning of new tle. ncer Res. 66, (26). 19. lin, G. A. & roce,. M. MicroRNA signtures in humn cncers. Nture Rev. ncer 6, (26). 2. O onnell, R. M., Tgnov, K. D., Boldin, M. P., heng, G. & Bltimore, D. MicroRNA-155 is induced during the mcrophge inflmmtory response. Proc. Ntl Acd. Sci. USA 14, (27). 21. Kto, T. et l. Efficient repliction of the genotype 2 heptitis virus sugenomic replicon. Gstroenterology 125, (23). 22. heng, G., Zhong, J. & hisri, F. V. Inhiition of dsrna-induced signling in heptitis virus-infected cells y NS3 protese-dependent nd -independent mechnisms. Proc. Ntl Acd. Sci. USA 13, (26). 23. Liu,. G. et l. An oligonucleotide microchip for genome-wide microrna profiling in humn nd mouse tissues. Proc. Ntl Acd. Sci. USA 11, (24). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk G. lin (OSU) for help with the microrry studies, D. Burton (TSRI) for E2-ntiodies, nd J. Zhong (TSRI) for mny discussions. This work ws supported y the NIH (I.M.P.), privte dontion from. Evns to F.V., nd the NIH nd USD Acdemic Sente Funding (M.D.). Author ontriutions G.. nd S.W. contriuted eqully to this work. Author Informtion Reprints nd permissions informtion is ville t orrespondence nd requests for mterils should e ddressed to M.D. (midvid@ucsd.edu) Nture Pulishing Group

5 doi:1.138/nture625 METHODS Trnsfection of mirna mimics nd nti-mirnas into Huh7 or Huh7 replicon cells. When JFH1-replicon-contining Huh7 cells (7% confluent) were used for trnsfection experiments, constnt totl mounts of mirna mimics or nti-mirnas (Dhrmcon nd Applied Biosystems, respectively) were comined with Mirus trnsfection regent (Mirus Bio/Fisher Scientific) ccording to the mnufcturers instructions nd dded to the cells (trnsfection efficiency.9%). Where indicted, IFN (DBL Biomedicl Lortories) ws dded to the cultures fter 8 h, nd cells were hrvested 48 h post trnsfection. Virl repliction ws determined y rel-time PR nlysis of virl genomic RNA. Similrly, for experiments using infectious HV, Huh7 cells were trnsfected with mirnas nd nti-mirnas, s descried ove. HV infection (multiplicity of infection 5.5) ws performed 8 h post trnsfection, nd IFN ws dded where indicted to the cultures t the sme time. ells were hrvested t the indicted time points during the phse of exponentil virl RNA mplifiction, nd virl repliction ws determined y rel-time PR nlysis of virl genomic RNA, or y determintion of the numer nd size of virl foci fter 72 h. J6F JFH1 chimeric HV genomes. Recominnt PR ws used to replce the J6F NS5B-39 UTR region in pv-j6f 24 with the corresponding sequences from JFH-1 present in pu-vjfh 25. First, using primers XJFH (GATTAG- AAGTTGATGTGAG), nd NS5Bup (TATGTATATTG- GAGGGGT) nd primers NS5Blo (GAGGGTAGGAGTATG- AATGGAG) nd XhoJ6F (AGGTTATTTTATGTG) the NS5B-39 UTR region of JFH-1 nd n NS5A-NS5B frgment contining unique XhoI restriction site from J6F were mplified, respectively. The two PR products were mutully extended nd mplified using the primers XhoJ6F nd XJFH, nd the resultnt PR product ws cloned into pgem-tesy (Invitrogen) yielding pte-j6/jfh, nd the insert ws verified y DNA sequencing. Finlly, the XhoI to XI frgment in pv-j6f ws replced y the 2.1-k XhoI/XI frgment excised from pte-j6/jfh, yielding pv-j6f/ JFHNS5B3 UTR contining the J6/JFH chimeric HV genome with the NS5B-39 UTR of J6F replced y the corresponding sequences from JFH-1. A similr recominnt PR pproch ws used to replicte the J6F JFH-1 chimeric HV genome Jc1 26 y replcing the corresponding JFH-1 core-ns2 region in pu-vjfh with the corresponding sequences from J6F to yield the plsmid pu-vjc1. Infectious JFH-1 nd chimeric viruses were produced y trnsfection of in vitro synthesized genomic HV RNA into Huh cells nd virus stocks contining focus forming units per ml (f.f.u. ml 21 ) were prepred s descried previously Yngi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. Heptitis virus: n infectious moleculr clone of second mjor genotype (2) nd lck of viility of intertypic 1 nd 2 chimers. Virology 262, (1999). 25. Wkit, T. et l. Production of infectious heptitis virus in tissue culture from cloned virl genome. Nture Med. 11, (25). 26. Pietschmnn, T. et l. onstruction nd chrcteriztion of infectious intrgenotypic nd intergenotypic heptitis virus chimers. Proc. Ntl Acd. Sci. USA 13, (26). 27. Zhong, J. et l. Roust heptitis virus infection in vitro. Proc. Ntl Acd. Sci. USA 12, (25). 27 Nture Pulishing Group