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Selective nuclear export of viral mRNAs in influenza-virus-infected cells
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Selective nuclear export of viral mRNAs in influenza-virus-infected cells Zhongying Chen and Robert M. Krug
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nfluenza virus is one of the The NS1A protein of influenza A virus are synthesized by the viral few RNA viruses to synthe- specifically inhibits the cellular machinery polymerase itself, which resize its mRNA in the nucleus that processes the 39 ends of cellular preiteratively copies a short of infected cells1. Viral mRNA mRNAs by targeting two of the essential stretch of U residues in the synthesis is catalysed by the proteins of this machinery. Because the template genome RNA during viral RNA-dependent RNA virus does not use this cellular machinery transcription6,7. polymerase, which is brought to synthesize the 39 poly(A) ends of viral into the nucleus as part of the mRNA, the nuclear export of cellular but Nuclear export of mRNA nucleocapsids of the virions not viral mRNAs is selectively inhibited. All mRNAs that are synthethat initiate infection. At subsized in the nucleus must be Z. Chen and R.M. Krug* are in the Institute for sequent times in the infection, exported to the cytoplasm and Molecular Biology, Section of Molecular newly synthesized polymerase Cellular for translation. Export occurs Genetics and Microbiology, The University of Texas complexes are transported to through nuclear pore comat Austin, 2500 Speedway, Austin, TX 78712, USA; the nucleus to function in viral plexes, large structures comZ. Chen is currently at Johnson & Johnson, Consumer Products Worldwide, 199 Grandview mRNA synthesis. posed of many different proRoad, Skillman, NJ 08558-9418, USA. The viral mRNAs that are teins called nucleoporins8. The *tel: 11 512 232 5563, synthesized in the nucleus nuclear export of cellular fax: 11 512 232 5565, undergo some of the same mRNAs is mediated by several e-mail: [email protected] processing steps as the cellular proteins that are bound to pre-mRNAs that are synthethem and their pre-mRNA sized by cellular RNA polymerase II (Ref. 1). The precursors9. The hnRNP proteins, abundant nuclear host nuclear splicing machinery is exploited to splice proteins that are bound to pre-mRNAs and mRNAs two full-length viral mRNAs, the NS1A and M1 in the nucleus, have been implicated in the nuclear mRNAs [encoding the non-structural (NS1A) and export of mRNAs. Many hnRNP proteins shuttle bematrix (M1) proteins, respectively]. The resulting tween the nucleus and cytoplasm. These proteins consmaller viral mRNAs encode two other proteins, NS2 tain amino acid sequences that function as nuclearand M2 (the viral ion-channel protein), respectively1. export signals, or NESs, which often differ from the Thus, the NS1A and M1 mRNAs function as both short leucine-rich sequence found in many other shutpre-mRNAs and mRNAs, and only a proportion tling proteins. For example, the NES of hnRNP A1 (~10%) of these mRNAs is spliced. Incomplete splic- is a 38 amino acid domain, called M9, which also ing also occurs in retrovirus-infected cells2. By con- functions as a nuclear-localization signal, or NLS trast, cellular pre-mRNAs that contain a single intron (Ref. 10). These hnRNP NESs can be ‘overridden’ by nuclear-retention signals, or NRSs, which are found are completely spliced. Although both cellular and influenza viral mRNAs in other hnRNP proteins11,12. The NRS-containing contain cap structures (m7 GpppNm) at their 59 ends hnRNP proteins must be removed from mRNA moland poly(A) tails at their 39 ends, the mechanisms by ecules before nuclear export can occur. Additionally, which these 59 and 39 sequences are acquired differ. several other proteins that are associated with nuclear The 59 cap structures of cellular mRNAs are synthe- cellular pre-mRNAs and mRNAs have been implisized de novo by cellular enzymes3, whereas the 59 cated in nuclear export. For example, TAP, the procap structures of influenza viruses are ‘snatched’ from tein that mediates the nuclear export of the mRNAs cellular pre-mRNAs during viral mRNA synthesis1 encoded by some retroviruses13,14, might also mediate (Fig. 1). An endonuclease that is intrinsic to the viral the nuclear export of cellular mRNAs9. polymerase cleaves cellular capped pre-mRNAs to Processing of cellular pre-mRNAs to form mature produce capped fragments 10–13 nucleotides (nt) in mRNAs is usually required for their export from the length, which serve as primers for viral mRNA syn- nucleus. The removal of introns by splicing is apparthesis. The mature 39 ends of cellular mRNAs are ently required as binding of splicing factors to intronproduced in two post-transcriptional steps: endonu- containing pre-mRNAs commits these pre-mRNAs cleolytic cleavage of the primary transcripts synthe- to spliceosome formation15. Most mature cellular sized by cellular RNA polymerase II, followed by mRNAs contain 39 poly(A) tails, the formation of polyadenylation of the upstream cleavage products4,5. which is required for mRNA export16,17. Positioning a By contrast, the 39 poly(A) tails of viral mRNAs transcribed poly(A) tract at the end of an mRNA by 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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ribozyme cleavage does not result (a) Primer production in efficient nuclear export17, indicating that the 39-processing 10–13 nt cellular pre-mRNA reaction itself, and not simply m7 G p p p N m N A pNpNp the presence of the poly(A) tail, is required for nuclear export. As G pNpNp m7 G p p p N m N described below, the results ob5¢ cap tained in influenza-virus-infected cells have verified this conclusion. Virion RNA (b) Initiation Cellular pre-mRNAs and mRNAs contain 59 cap structures bound UpCpGpUpUpUpUpCp 10–13 nt to a complex of two proteins18,19. These cap structures and their asG p sociated proteins are not required p A m7 G p p p N m N for the nuclear export of cellular p mRNAs, but do stimulate such export. (c) Elongation Virion RNA In cells infected by influenza A virus, the nuclear export of celluUpCpGpUpUpUpUpCp lar mRNAs is blocked, and cellu7 G p p p N m N m ApGpCpApApApApGp lar pre-mRNAs and mRNAs are 1,20 degraded in the nucleus . This Viral mRNA trends in Microbiology block in nuclear export is selective: all viral mRNAs are effiFig. 1. ‘Cap-snatching’ by the influenza viral polymerase. The viral polymerase is incapable of iniciently exported21. Consequently, tiating viral mRNA synthesis unless a primer is supplied; cellular pre-mRNAs and mRNAs in the nucleus of infected cells are ‘cannibalized’ to supply these primers1. (a) An endonuclease that is viral mRNAs are the predomiintrinsic to the polymerase cleaves capped (m7 GpppNm-containing) cellular nuclear RNAs 10–13 nant newly synthesized mRNAs nucleotides (nt) from their 59 ends, preferentially at a purine residue. The resulting capped fragthat reach the translation maments serve as primers for the initiation of viral mRNA synthesis. (b) Transcription is initiated by chinery in the cytoplasm of inthe incorporation of a G residue onto the 39 end of the resulting fragments, directed by the penultimate C residue of the genome RNAs. (c) Influenza viral mRNA synthesis requires continuous fected cells, thereby contributing synthesis of cellular pre-mRNAs to provide primers for the initiation of viral mRNA chains. to the shutdown of host-cell gene expression and the selective synthesis of viral proteins. Other mechanisms must also operate to mediate this selec- to double-stranded (ds) RNA and a specific stem tive synthesis, because the cytoplasm contains many bulge in U6 small nuclear (sn) RNA31–33. NS1A exists cellular mRNAs that are synthesized prior to infec- as a dimer in vivo and in vitro, and the RNA-binding tion. Although these cellular mRNAs are stable and domain is also required for this dimerization34. The functional (as assayed by their ability to direct trans- RNA-binding and dimerization domain comprises lation in in vitro systems), their translation is selec- the amino-terminal 73 amino acids, which, in vitro, tively inhibited in infected cells by mechanisms that can function independently of the rest of the prohave not yet been elucidated20,22,23. It has been reported tein35. Nuclear magnetic resonance and X-ray crystalthat cellular mRNAs are degraded in the cytoplasm of lography have demonstrated that, in the absence of infected cells20,24,25, but this degradation occurs subse- RNA, this dimeric RNA-binding domain adopts a quent to the block in their translation20,22. novel six-helical-chain fold36–38. A second functional domain, which is located in the carboxyl half of the NS1A and the inhibition of nuclear export molecule, is not required for RNA binding, but is The NS1 protein of influenza A virus (NS1A) was nonetheless required for the inhibition of the nuclear identified in 1971 (Ref. 26), but little progress in elu- export of poly(A)-containing mRNAs29. It was postucidating its function was made until the early 1990s. lated that this domain is the effector domain that inTransient transfection experiments established that teracts with host nuclear proteins to inhibit nuclear one of its functions is the inhibition of the nuclear RNA export35, a claim which was subsequently subexport of poly(A)-containing spliced mRNAs, that stantiated. Although mutagenesis originally identified is, mRNAs without introns27–30. Nuclear export of residues 134–161 as the effector domain35, it is likely mRNAs is inhibited only when they contain a poly(A) that it extends from residue 74 (at the end of the tail produced by the cellular cleavage/polyadenyl- RNA-binding domain) to residue 237, the carboxyation system; NS1A does not inhibit the nuclear ex- terminal amino acid. port of histone mRNA, the 39 end of which is produced by a different set of factors28,30. Selective inhibition of nuclear export Mutational analysis has identified two functional CPSF and PABII domains in this 237-residue protein29. A sequence NS1A specifically inhibits the cellular 39-end processnear the amino end comprises the RNA-binding do- ing machinery by targeting two proteins: the human main, which binds with similar dissociation constants 30-kDa subunit of the cleavage and polyadenylation
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A AUA A A
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Fig. 2. The mature 39 ends of cellular mRNAs are generated by endonucleolytic cleavage of the pre-mRNAs, followed by polyadenylation of the upstream cleavage product4,5. A minimum of two sequence elements define a poly(A) site: (1) the almost invariant AAUAAA sequence, 10–30 nucleotides upstream of the cleavage site; and (2) a variable U- or G/Urich element (DSE) located 20–40 nucleotides downstream of the cleavage site. Cleavage and poly(A) addition are carried out by a large complex of multisubunit proteins. The cleavage and polyadenylation specificity factor (CPSF) binds to the AAUAAA sequence in the pre-mRNA via its 160kDa subunit and possibly its 30-kDa subunit. Cleavage stimulation factor (CstF) binds to the DSE and stabilizes the binding of CPSF to the premRNA. Cleavage factors I and II (CFI and CFII) are also required for cleavage. Poly(A) polymerase (PAP) is also detected in the cleavage complex. After cleavage of the pre-mRNA, three factors – CstF, CFI and CFII – leave the complex, and PAP catalyses the addition of a short (~10 nucleotides) poly(A) tail. Processive elongation of the poly(A) tail requires an additional protein, poly(A)-binding protein II (PABII)43,45. PABII binds poly(A) with high affinity and specificity and, along with CPSF, tethers PAP to the RNA substrate, thereby facilitating processive synthesis of a long poly(A) tail. Cellular mRNAs containing elongated poly(A) tails are exported from the nucleus.
specificity factor (CPSF)39 and poly(A)-binding protein II (PABII)40. It is likely that these proteins bind to non-overlapping regions of the NS1A protein effector domain, and can therefore bind NS1A simultaneously40.
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These two cellular factors have different functions (Fig. 2). CPSF binds to the AAUAAA poly(A) signal located 10–30 nt upstream of the cleavage site in premRNAs and is required for both cleavage and polyadenylation of pre-mRNAs4,41. The 30-kDa subunit is one of the two CPSF subunits that have been implicated in the specific binding to the AAUAAA polyadenylation signal41,42. By contrast, PABII functions after 39 cleavage of the cellular premRNA, and is required for the processive elongation of poly(A) chains, which is catalysed by the cellular poly(A) polymerase (PAP)5,41. After PAP adds a short poly(A) tail of ~10 nt to the cleaved pre-mRNA, PABII, along with CPSF, tethers PAP to the RNA substrate, thereby facilitating processive synthesis of a long poly(A) tail in a single rapid step43. In the absence of PABII, only short poly(A) tails are added to the 39-cleaved pre-mRNA. Several lines of evidence have established that the binding of influenza virus NS1A to the 30-kDa CPSF subunit dramatically inhibits CPSF function and hence inhibits the 39-end cleavage and polyadenylation of cellular pre-mRNAs39. Immunoprecipitation experiments have shown that, in influenza-virusinfected cells, NS1A is physically associated with the 30-kDa CPSF subunit, and that this subunit is present in CPSF complexes containing all four essential protein subunits39. In vitro assays established that binding of the viral NS1A protein to the 30-kDa subunit prevents CPSF binding to the RNA substrate and inhibits 39-end cleavage and polyadenylation of cellular pre-mRNAs39. Transfection experiments demonstrated that NS1A inhibits the 39 cleavage and polyadenylation of pre-mRNAs in vivo and that the uncleaved pre-mRNAs remain in the nucleus39. Finally, influenza viruses that contain temperature-sensitive mutations in the NS1A protein-coding sequence inhibit 39 cleavage of cellular pre-mRNAs at the permissive, but not at the non-permissive, temperature44. Because NS1A inhibits the 39 cleavage of cellular pre-mRNAs, it came as a surprise that it also inhibits a reaction that occurs after cleavage, namely the processive elongation of poly(A) chains mediated by the PABII (Ref. 40). Several experiments definitively established that NS1A inhibits PABII function40. In vitro, NS1A binds PABII and inhibits its ability to stimulate the processive synthesis of long poly(A) tails catalysed by PAP. NS1A does not inhibit the binding of PABII to poly(A), and instead blocks the functional interaction of PABII with PAP. In influenza-virus-infected cells, 39 cleavage of some cellular pre-mRNAs still occurs despite the inhibition of CPSF function, followed by the addition of short poly(A) tails catalysed by the cellular PAP. These premRNAs accumulate in the nucleus of infected cells. Subsequent processive elongation of these short poly(A) tails does not occur because NS1A inhibits the function of the cellular PABII protein40. Two-pronged attack The two-pronged attack of NS1A against the cellular 39-end-processing machinery is depicted in Fig. 3.
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Fig. 3. The two-pronged mechanism by which NS1A inhibits the cellular 39-end-processing system in influenza-virus-infected cells. Pathway I: the binding of the NS1 protein to the 30-kDa subunit of CPSF blocks the binding of CPSF to the AAUAAA sequence of some cellular pre-mRNA molecules, thereby blocking 39 cleavage of these pre-mRNAs39. The uncleaved pre-mRNAs remain in the nucleus. Pathway II: CPSF binds to the AAUAAA sequence of other cellular pre-mRNA molecules, despite the binding of the NS1A protein to the 30-kDa CPSF subunit. A short poly(A) sequence is then added to these cleaved pre-mRNAs by PAP in a CPSF-dependent reaction. Subsequent elongation of the short poly(A) sequence is blocked by the binding of the NS1A protein to the PABII protein, resulting in the nuclear accumulation of cleaved pre-mRNAs containing short poly(A) tails40. In both pathways, individual NS1A protein molecules form complexes with both PABII and the 30 kDa subunit of CPSF. In these complexes the two cellular 39 processing proteins, 30 kDa CPSF and PABII, also bind directly to each other. Both species of the cellular pre-mRNAs that are sequestered in the nucleus are cleaved by the viral cap-dependent endonuclease to produce the primers required for viral mRNA synthesis. The 39 terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of 4–7 Us in the virion RNA templates. The poly(A)-containing viral mRNAs are exported from the nucleus. Reproduced, with permission, from Ref. 60. Abbreviations: CPSF, cleavage and polyadenylation specificity factor; NS1A, non-structural 1A; PAB, poly(A)-binding protein; PAP, poly(A) polymerase.
Each NS1A protein molecule in influenza-virusinfected cells forms a complex with both PABII and the 30-kDa CPSF subunit. Because it has been found that PABII and the 30-kDa CPSF subunit also bind to each other in vitro40, it is likely that all three proteins form a ternary complex. Consequently, in Fig. 3, PABII is presumed to be in the 39-processing complexes that are formed prior to 39 cleavage of the premRNA. As a result of the formation of the complex between the 30-kDa CPSF subunit and NS1A, the binding of CPSF to the AAUAAA sequence of some cellular pre-mRNA molecules is blocked, so that 39 cleavage and the subsequent addition of A residues does not occur39 (pathway I). Other cellular premRNA molecules continue to be cleaved to a certain extent in influenza-virus-infected cells, indicating that NS1A does not completely inhibit the functional binding of CPSF to the AAUAAA sequence (pathway II). It is possible that the cleavage stimulation factor (CstF), which binds to a U- or G/U-rich element
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downstream of the cleavage site4,5, might mediate formation of a 39-end-processing complex that is not effectively blocked by NS1A. Because CstF binding stabilizes the interaction of CPSF with the AAUAAA sequence4,5, the presence of a downstream sequence that affords optimal binding of CstF might be expected to offset the NS1A-mediated inhibition of CPSF binding (see Fig. 2). After the pre-mRNAs are cleaved, a short poly(A) sequence is added by PAP in a reaction that requires CPSF. Subsequent elongation of the short poly(A) sequence is blocked because NS1A inhibits PABII function40, which, along with CPSF, is required to tether PAP to poly(A) so it can catalyse the processive addition of A residues43,45. Consequently, via a two-pronged attack against PABII and CPSF, NS1A efficiently blocks the 39-end processing of cellular pre-mRNAs in influenza-virusinfected cells. Neither the uncleaved cellular pre-mRNAs (produced in pathway I) nor the cleaved cellular pre-mRNAs
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Box 1. Other viruses that regulate the nuclear export of mRNA • HIV-1: the HIV-1 Rev protein mediates the nuclear export of unspliced and partially spliced viral mRNAs, overriding their nuclear retention via spliceosome formationa. • Adenovirus: a complex of two viral proteins [E1B (55 kDa) and E4 open reading frame (ORF) 6] mediates the selective nuclear export of viral mRNAsb. • Herpes simplex virus 1: the viral ICP27 protein promotes the export of viral mRNAs, which, unlike the vast majority of cellular mRNAs, are synthesized as colinear mRNAs lacking intronsc,d. References a Cullen, B.R. (1995) Regulation of HIV gene expression. AIDS 9, S19–S32 b Dobbelstein, M. et al. (1997) Nuclear export of the E1B 55-kDa and E4 34kDa adenoviral oncoproteins mediated by a rev-like signal sequence. EMBO J. 16, 4276–4284 c Phelan, A. et al. (1996) Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts. J. Virol. 70, 5255–5265 d Sandri-Goldin, R.M. (1998) ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 6, 868–879
containing short poly(A) tails (produced in pathway II) are exported from the nucleus39,40. These observations not only confirm previous results indicating that 39-end processing of pre-mRNAs is required for their nuclear export16,17, but also indicate that nuclear export of pre-mRNAs occurs only after the PABII-mediated elongation of poly(A) chains is accomplished. By blocking the nuclear export of cellular mRNAs, the influenza viral NS1A protein inhibits the expression of cellular mRNAs synthesized after infection. In addition, the cellular pre-mRNAs and mRNAs that are trapped in the nucleus are accessible to the viral cap-dependent endonuclease for the production of the capped RNA primers that are required for viral mRNA synthesis1 (Figs 1,3). Because of the loss of their 59-cap structures, the sequestered cellular RNAs should become susceptible to nuclease digestion46 and in fact, it has been shown that cellular pre-mRNAs and mRNAs are degraded in the nucleus of infected cells20. Another mechanism The binding of the viral NS1A protein to the cellular PABII protein could also inhibit the nuclear export of cellular mRNAs by another mechanism. PABII protein molecules shuttle between the nucleus and the cytoplasm, an activity that implicates this protein in the nuclear export of cellular mRNAs40,47. The hypothesis that PABII plays an essential role in the nuclear export of cellular mRNAs is consistent with
Questions for future research • What is the mechanism by which influenza viral mRNAs are exported from the nucleus of infected cells? • Does the PABII protein participate in the nuclear export of cellular poly(A)-containing mRNAs? • What amino acid sequences of the influenza virus NS1A protein interact with the cellular 30-kDa CPSF and PABII proteins, and are similar interacting sequences found in cellular proteins?
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the finding that the 39-processing reaction, rather than simply the presence of a 39 poly(A) tail, is required for the nuclear export of cellular mRNAs16,17. It can be argued that the reason that the 39-end processing of cellular pre-mRNAs is required16,17,39,40 is that the elongated poly(A) tails bind PABII protein molecules that play an important role in mediating nuclear export. As discussed above, many other proteins that are associated with cellular pre-mRNAs and mRNAs in the nucleus have also been implicated in the nuclear export of cellular mRNAs. One possibility is that the participation of a large group of proteins in the nuclear export of mRNAs ensures that only mature, fully processed mRNAs are exported9. The presumption is that the association of these proteins with pre-mRNAs is linked to the acquisition of features of mature mRNA molecules during nuclearprocessing events. PABII would certainly link nuclear export with a processing step that is required for the production of mature mRNAs. Because NS1A inhibits the nuclear export of PABII protein molecules40, PABII would be incapable of functioning in the nuclear export of any mature cellular mRNAs that are produced despite the NS1A-mediated inhibition of 39-end processing. (See Box 1 for other viruses that regulate the nuclear export of mRNA.) Roles of the NS1A effector domain Several lines of evidence have established that the NS1A effector domain is required for the inhibition of 39-end processing of cellular pre-mRNAs. Both the 30-kDa CPSF subunit and PABII bind to the effector domain, and its deletion eliminates the ability of NS1A to inhibit 39 poly(A) processing39,40. A requirement for the RNA-binding domain, as well as the effector domain, in the inhibition of 39 poly(A) processing was suggested by early mutagenesis experiments, which indicated that mutations in the RNA-binding domain, as well as in the effector domain, result in the loss of the ability of the NS1A protein to inhibit the nuclear export of cellular mRNAs29. However, the mutant NS1A proteins used in these studies are inactive in both RNA binding and dimerization34,38. Because the loss of dimerization is a profound change in the structure of NS1A, the effector domain, as well as the RNA-binding domain, might be inactivated in such mutant proteins. To avoid this pitfall, a different mutant NS1A protein was designed based on the 3-D structure of the RNA-binding domain. This mutant lacks RNA-binding activity but retains its dimeric structure38. Because this mutant protein retains its ability to inhibit the 39-end processing of cellular premRNAs40 (Y. Li et al., submitted), it was concluded that the RNA-binding domain is not required for this inhibition, and that the effector domain alone is responsible for inhibiting 39-end processing of cellular pre-mRNAs and hence the nuclear export of cellular mRNAs. All naturally occurring influenza A viruses examined to date encode an NS1A protein that contains an effector domain48. At one time it was thought that one naturally occurring influenza A virus, A/Turkey/
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Oregon/71, encoded an NS1A protein that lacked the carboxy-terminal sequence containing the effector domain49. However, it has now been established that naturally occurring A/Turkey/Oregon/71 encodes a full-length NS1A protein48, indicating that the previously characterized virus isolate was probably generated during multiple passages in the laboratory. Consequently, the effector domain function of NS1A is conserved in nature, that is, under conditions of natural selection, indicating that this function probably plays important and necessary roles in natural infections. These necessary roles have not yet been fully delineated. The inhibition of the nuclear export of cellular mRNAs, which enhances viral gene expression, could make the virus more pathogenic and virulent in natural infections. In addition, the effector domain has one other known activity that is likely to be crucial for virus replication: an NES that is activated during infection50. However, the role of this NES in virus replication has not yet been determined. Furthermore, the effector domain might have other essential functions that have not yet been elucidated. For example, laboratory-generated viruses that lack large deletions of the effector domain fail to protect the virus against IFN (Ref. 51), indicating that the effector domain, as well as the RNA-binding domain31,32,52, might be required to protect the virus against IFN. Only in Vero cells, which are defective in IFN production, do these mutant viruses grow as well as wild-type virus in tissue culture51. The efficiency of replication of these viruses in MDCK cells, which produce IFN, is inversely correlated with the length of the effector domain of the encoded NS1A protein, and these viruses are attenuated in wild-type mice that produce IFN (Ref. 51). The requirement of the NS1A effector domain for replication in tissue culture cells has also been established by the existence of at least one mutation in the sequence of the effector domain that results in a temperature-sensitive block in virus replication44. Interestingly, although the RNA-binding domain of the NS1 protein encoded by influenza B virus (NS1B protein) possesses the same RNA-binding activities as NS1A, the NS1B effector domain does not function in the inhibition of the nuclear export of cellular mRNAs53. Consequently, this function must not be required for the propagation of influenza B viruses in nature, and the function(s) of the large carboxy-terminal region of NS1B proteins, which is encoded by all naturally occurring influenza B viruses49, is unknown. This fundamental difference between the effector domains of the NS1A and NS1B proteins probably contributes to the different biological properties of influenza A and B viruses. Conclusions and remaining issues Influenza virus NS1A blocks the nuclear export of cellular mRNAs by binding to, and inhibiting the function of, two cellular proteins that are required for the 39-end processing of cellular pre-mRNAs39,40. The inhibition of the cellular 39-end-processing machinery should not block the formation of the 39 poly(A)
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tails of viral mRNAs because the tails are produced by the viral polymerase, and not by the cellular 39-end processing machinery6,7. In fact, the influenza viral mRNAs in infected cells possess 39 poly(A) tails of 150–200 bases54. However, although the presence of elongated poly(A) tails at the 39 ends of influenza viral mRNAs is necessary for nuclear export7, it is unlikely to be sufficient. As is the case for cellular mRNAs, the nuclear export of influenza viral mRNAs is presumably mediated by one or more proteins that are bound to these mRNAs. The identity of such proteins, which could be cellular and/or virus-specific, is unknown. Because there are three different types of influenza virus mRNAs, more than one mechanism of nuclear export might operate in virus-infected cells. Of the eight influenza virus genome RNA segments, the five largest are transcribed into monocistronic mRNAs that lack introns. Only a small number of cellular genes, including histone genes, are transcribed into colinear mRNAs that lack introns55,56. It has been shown that efficient nuclear export of one of these cellular mRNAs, poly(A)-containing histone H2a mRNA, is mediated by a specific sequence in the mRNA57,58. The second type of influenza viral mRNA is encoded by the two smallest influenza virus genome segments (the M1 and NS1A mRNAs), which are transcribed into intron-containing mRNAs. Only a small proportion (~10%) of these two mRNAs is spliced, and the unspliced molecules are efficiently exported from the nucleus to be translated1. Incomplete splicing and the nuclear export of unspliced mRNAs also occurs in retrovirus-infected cells2. The nuclear export of unspliced mRNAs encoded by some retroviruses is mediated by TAP, a cellular protein that recognizes an RNA sequence in the unspliced retroviral mRNAs13,14. Like these retroviruses, influenza virus does not encode a viral protein that mediates the nuclear export of its unspliced viral mRNAs59. The viral M2 and NS2 viral mRNAs that are produced by splicing comprise the third type of influenza viral mRNA. The nuclear export of these spliced mRNAs might be similar to that of their cellular spliced mRNA counterparts, except that components of the cellular 39-end-processing system would not participate in the nuclear export of the viral M2 and NS2 mRNAs. Clearly, not much is known about the mechanism of the nuclear export of the three types of influenza viral mRNAs. Acknowledgements The work carried out in the authors’ laboratory is supported by National Institutes of Health grant AI 11772 to R.M.K. References 1 Krug, R.M. et al. (1989) Expression and replication of the influenza virus genome. In The Influenza Viruses (Krug, R.M., ed.), pp. 89–152, Plenum Press 2 O’Reilly, M.M. et al. (1995) Two strong 59 splice sites and competing, suboptimal 39 splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. Virology 213, 373–385 3 Shuman, S. (1995) Capping enzyme in eukaryotic mRNA synthesis. Prog. Nucleic Acid Res. Mol. Biol. 50, 101–129
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4 Colgan, D.F. and Manley, J.L. (1997) Mechanism and regulation of mRNA polyadenylation. Genes Dev. 11, 2755–2766 5 Wahle, E. and Kuhn, U. (1997) The mechanism of 39 cleavage and poladenylation of eukaryotic pre-mRNA. Prog. Nucleic Acid Res. Mol. Biol. 57, 41–71 6 Robertson, J.S. et al. (1981) Polyadenylation sites for influenza virus mRNA. J. Virol. 38, 157–163 7 Poon, L.L. et al. (1999) 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. 73, 3473–3476 8 Stoffler, D. et al. (1999) The nuclear pore complex: from molecular architecture to functional dynamics. Curr. Opin. Cell Biol. 11, 391–401 9 Nakielny, S. and Dreyfuss, G. (1999) Transport of proteins and RNAs in and out of the nucleus. Cell 99, 677–690 10 Pollard, V.W. et al. (1996) A novel receptor-mediated nuclear protein import pathway. Cell 86, 985–994 11 Pinol-Roma, S. and Dreyfuss, G. (1992) Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 350, 730–732 12 Nakielny, S. and Dreyfuss, G. (1996) The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals. J. Cell Biol. 134, 1365–1373 13 Pasquinelli, A.E. et al. (1997) The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16, 7500–7510 14 Gruter, P. et al. (1998) TAP, the human homolog of mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, 649–659 15 Legrain, P. and Rosbash, M. (1989) Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57, 573–583 16 Eckner, R. et al. (1991) Mature mRNA 39 end formation stimulates RNA export from the nucleus. EMBO J. 10, 3513–3522 17 Huang, Y. and Carmichael, G. (1996) Role of polyadenylation in nucleocytoplasmic transport of mRNA. Mol. Cell. Biol. 16, 1534–1542 18 Izaurralde, E. et al. (1995) A cap-binding protein complex mediating U snRNA export. Nature 376, 709–712 19 Visa, N. et al. (1996) A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J. Cell Biol. 133, 5–14 20 Katze, M.G. and Krug, R.M. (1984) Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells. Mol. Cell. Biol. 4, 2198–2206 21 Shapiro, G.I. et al. (1987) Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs. J. Virol. 61, 764–773 22 Katze, M.G. et al. (1986) Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus. J. Virol. 60, 1027–1039 23 Katze, M.G. and Krug, R.M. (1990) Translational control in influenza virus-infected cells. Enzyme 44, 265–277 24 Inglis, S.C. (1982) Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus. Mol. Cell. Biol. 1644–1648 25 Beloso, A. et al. (1992) Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff. J. Gen. Virol. 73, 575–581 26 Lazarowitz, S.G. et al. (1971) Influenza virus structural and nonstructural proteins in infected cells and their plasma membranes. Virology 46, 830–843 27 Alonso-Caplen, F.V. et al. (1992) Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev. 6, 255–267 28 Fortes, P. et al. (1994) Influenza virus NS1 protein inhibits
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pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 13, 704–712 Qian, X.Y. et al. (1994) Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA. J. Virol. 68, 2433–2441 Qiu, Y. and Krug, R.M. (1994) The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J. Virol. 68, 2425–2432 Hatada, E.R. and Fukuda, R. (1992) Binding of influenza A virus NS1 protein to dsRNA in vitro. J. Gen. Virol. 73, 3325–3329 Lu, Y. et al. (1995) Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase (PKR) that phosphorylates the eIF-2 translation initiation factor. Virology 214, 222–228 Qiu, Y. et al. (1995) The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6–U2 and U6–U4 snRNA interactions during splicing. RNA 1, 304–316 Nemeroff, M.E. et al. (1995) The influenza virus NS1 protein forms multimers in vitro and in vivo. Virology 212, 422–428 Qian, X. et al. (1995) An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA 1, 948–956 Chien, C. et al. (1997) A novel RNA-binding motif in influenza A virus non-structural protein 1. Nat. Struct. Biol. 4, 891–895 Liu, J. et al. (1997) Crystal structure of the unique multifunctional RNA-binding domain of the influenza virus NS1 protein. Nat. Struct. Biol. 4, 896–899 Wang, W. et al. (1999) RNA-binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5, 195–205 Nemeroff, M. et al. (1998) Influenza virus NS1 protein interacts with the 30kd subunit of cleavage and specificity factor and inhibits 39 end formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000 Chen, Z. et al. (1999) Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 39 end processing machinery. EMBO J. 18, 2273–2283 Barabino, S.M. and Keller, W. (1999) Last but not least: regulated poly(A) tail formation. Cell 99, 9–11 Barabino, S.M.L. et al. (1997) The 30-kd subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins. Genes Dev. 11, 1703–1716 Bienroth, S. et al. (1993) Assembly of a processive messenger RNA polyadenylation complex. EMBO J. 12, 585–594 Shimuzu, K. et al. (1999) Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology 254, 213–219 Wahle, E. and Keller, W. (1996) The biochemistry of polyadenylation. Trends Biochem. Sci. 21, 247–250 Furuichi, Y. et al. (1977) 59-terminal structure and mRNA stability. Nature 266, 235–239 Calado, A. et al. (2000) Deciphering the cellular pathway for transport of poly(A)-binding protein II. RNA 6, 245–256 Suarez, D.L. and Purdue, M.L. (1998) Multiple alignment comparison of the non-structural genes of influenza A viruses. Virus Res. 54, 59–69 Norton, G.P. et al. (1987) Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides. Virology 156, 204–213 Li, Y. et al. (1998) Regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus NS1 protein. Proc. Natl. Acad. Sci. U. S. A. 95, 4864–4869 Egorov, A. et al. (1998) Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J. Virol. 72, 6437–6441 Hatada, E. et al. (1999) Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells. J. Virol. 73, 2425–2433 Wang, W. and Krug, R.M. (1996) The RNA-binding and effector
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domains are conserved to different extents among influenza A and B viruses. Virology 223, 41–50 Plotch, S.J. and Krug, R.M. (1977) Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J. Virol. 21, 24–34 Kedes, L.H. (1979) Histone genes and histone messengers. Annu. Rev. Biochem. 48, 837–870 Nagata, S. et al. (1980) The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature 287, 401–408 Huang, Y. and Carmichael, G.G. (1997) The mouse histone H2a gene contains a small element that facilitates cytoplasmic
A well-targeted textbook Biochemistry and Molecular Biology of Antimicrobial Drug Action (5th edn) by T.J. Franklin and G.A. Snow Kluwer Academic Publishers, 1998. (£69.00/$122.13 hbk) (ix 1 166 pages) ISBN 0 412 82200 8
T
his book is essentially an updated, revised edition of the earlier fourth edition entitled Biochemistry of Antimicrobial Action. Its origins can therefore be traced to the 1970s when the first edition was published. Although there has been considerable growth in information on antimicrobial agents in the three decades since the first edition was published, the book remains admirably well focused and concise and is of comparable length to earlier editions. The broad layout of the current edition is similar to that of the fourth edition, with chapters describing the historical development of antimicrobial agents, the mechanisms of action and uptake of the principal drug classes, and the genetic and biochemical basis of resistance to antimicrobial agents. The book is written from a broad perspective as it deals with antibacterial, antifungal, antiviral and antiprotozoal agents. The principal antimycobacterial agents are also presented within the relevant
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accumulation of intronless gene transcripts and of unspliced HIV1-related mRNAs. Proc. Natl. Acad. Sci. U. S. A. 94, 10104–10109 58 Huang, Y. et al. (1999) Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. EMBO J. 18, 1642–1652 59 Alonso-Caplen, F.V. and Krug, R.M. (1991) Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rate of splicing and of the nucleocytoplasmic transport of NS1 mRNA. Mol. Cell. Biol. 11, 1092–1098 60 Lamb, R.A. and Krug, R.M. Orthomyxoviridae: the viruses and their replication. In Fields Virology (4th edn) (Knipe, D.M. and Howley, P.M., eds) Lippincott, Williams and Wilkins (in press)
target-based sections, for example, isoniazid and ethambutol are discussed in the chapter that deals with agents interfering with microbial cell wall biosynthesis. Antiseptics are also included within the definition of antimicrobial drugs. The clinical applications of the agents described are only referred to in broad terms and therefore readers wishing to gain specific information on the indications for use of a particular drug to treat disease will need to consult other texts. The previous edition of this book, somewhat confusingly for a text on antimicrobial agents, contained information on anticancer drugs; however, these descriptions have been omitted in the fifth edition, which therefore has a content more in-keeping with its title. The fifth edition now includes the phrase ‘molecular biology’ in its title to emphasize the important contributions made by molecular studies to the topic of antimicrobial agents. It is in this area that the book differs significantly from earlier editions by, for example, presenting X-ray crystallographic analysis of various bacterial blactamases and molecular descriptions of ribosome structure and multi-drug efflux systems in Gram-negative bacteria. Nevertheless, despite the comprehensive coverage of antimicrobial agents there are some important omissions. For example, neither the oxazolidinones, which represent an important new class of bacterial protein-synthesis inhibitors,
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nor the glycylcyclines (third-generation tetracycline analogues) are mentioned. Furthermore, for a book which stresses the importance of molecular biology for antimicrobial research, the significance and future impact of the extensive microbial genomesequencing projects on antimicrobial drug discovery is given only the very briefest of treatment. Despite the above criticisms, this book will be an important source of information for all those with an interest in antimicrobial agents including students, lecturers, laboratory-based molecular biologists and clinicians. However, its greatest value is likely to be as a core undergraduate textbook supporting those courses dealing with the molecular basis of antimicrobial drug action and resistance. Thus, undergraduate students will particularly benefit from the clearly written text, useful illustrations, chemical structures of drugs, and references for further reading provided at the end of each chapter. The book is also well indexed. It will certainly become a recommended text in those first and second year undergraduate modules on antibiotics that I currently teach within my own University. Ian Chopra Divn of Microbiology and Antimicrobial Research Centre, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK LS2 9JT
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Selective nuclear export of viral mRNAs in influenza-virus-infected cells Zhongying Chen and Robert M. Krug
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nfluenza virus is one of the The NS1A protein of influenza A virus are synthesized by the viral few RNA viruses to synthe- specifically inhibits the cellular machinery polymerase itself, which resize its mRNA in the nucleus that processes the 39 ends of cellular preiteratively copies a short of infected cells1. Viral mRNA mRNAs by targeting two of the essential stretch of U residues in the synthesis is catalysed by the proteins of this machinery. Because the template genome RNA during viral RNA-dependent RNA virus does not use this cellular machinery transcription6,7. polymerase, which is brought to synthesize the 39 poly(A) ends of viral into the nucleus as part of the mRNA, the nuclear export of cellular but Nuclear export of mRNA nucleocapsids of the virions not viral mRNAs is selectively inhibited. All mRNAs that are synthethat initiate infection. At subsized in the nucleus must be Z. Chen and R.M. Krug* are in the Institute for sequent times in the infection, exported to the cytoplasm and Molecular Biology, Section of Molecular newly synthesized polymerase Cellular for translation. Export occurs Genetics and Microbiology, The University of Texas complexes are transported to through nuclear pore comat Austin, 2500 Speedway, Austin, TX 78712, USA; the nucleus to function in viral plexes, large structures comZ. Chen is currently at Johnson & Johnson, Consumer Products Worldwide, 199 Grandview mRNA synthesis. posed of many different proRoad, Skillman, NJ 08558-9418, USA. The viral mRNAs that are teins called nucleoporins8. The *tel: 11 512 232 5563, synthesized in the nucleus nuclear export of cellular fax: 11 512 232 5565, undergo some of the same mRNAs is mediated by several e-mail: [email protected] processing steps as the cellular proteins that are bound to pre-mRNAs that are synthethem and their pre-mRNA sized by cellular RNA polymerase II (Ref. 1). The precursors9. The hnRNP proteins, abundant nuclear host nuclear splicing machinery is exploited to splice proteins that are bound to pre-mRNAs and mRNAs two full-length viral mRNAs, the NS1A and M1 in the nucleus, have been implicated in the nuclear mRNAs [encoding the non-structural (NS1A) and export of mRNAs. Many hnRNP proteins shuttle bematrix (M1) proteins, respectively]. The resulting tween the nucleus and cytoplasm. These proteins consmaller viral mRNAs encode two other proteins, NS2 tain amino acid sequences that function as nuclearand M2 (the viral ion-channel protein), respectively1. export signals, or NESs, which often differ from the Thus, the NS1A and M1 mRNAs function as both short leucine-rich sequence found in many other shutpre-mRNAs and mRNAs, and only a proportion tling proteins. For example, the NES of hnRNP A1 (~10%) of these mRNAs is spliced. Incomplete splic- is a 38 amino acid domain, called M9, which also ing also occurs in retrovirus-infected cells2. By con- functions as a nuclear-localization signal, or NLS trast, cellular pre-mRNAs that contain a single intron (Ref. 10). These hnRNP NESs can be ‘overridden’ by nuclear-retention signals, or NRSs, which are found are completely spliced. Although both cellular and influenza viral mRNAs in other hnRNP proteins11,12. The NRS-containing contain cap structures (m7 GpppNm) at their 59 ends hnRNP proteins must be removed from mRNA moland poly(A) tails at their 39 ends, the mechanisms by ecules before nuclear export can occur. Additionally, which these 59 and 39 sequences are acquired differ. several other proteins that are associated with nuclear The 59 cap structures of cellular mRNAs are synthe- cellular pre-mRNAs and mRNAs have been implisized de novo by cellular enzymes3, whereas the 59 cated in nuclear export. For example, TAP, the procap structures of influenza viruses are ‘snatched’ from tein that mediates the nuclear export of the mRNAs cellular pre-mRNAs during viral mRNA synthesis1 encoded by some retroviruses13,14, might also mediate (Fig. 1). An endonuclease that is intrinsic to the viral the nuclear export of cellular mRNAs9. polymerase cleaves cellular capped pre-mRNAs to Processing of cellular pre-mRNAs to form mature produce capped fragments 10–13 nucleotides (nt) in mRNAs is usually required for their export from the length, which serve as primers for viral mRNA syn- nucleus. The removal of introns by splicing is apparthesis. The mature 39 ends of cellular mRNAs are ently required as binding of splicing factors to intronproduced in two post-transcriptional steps: endonu- containing pre-mRNAs commits these pre-mRNAs cleolytic cleavage of the primary transcripts synthe- to spliceosome formation15. Most mature cellular sized by cellular RNA polymerase II, followed by mRNAs contain 39 poly(A) tails, the formation of polyadenylation of the upstream cleavage products4,5. which is required for mRNA export16,17. Positioning a By contrast, the 39 poly(A) tails of viral mRNAs transcribed poly(A) tract at the end of an mRNA by 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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ribozyme cleavage does not result (a) Primer production in efficient nuclear export17, indicating that the 39-processing 10–13 nt cellular pre-mRNA reaction itself, and not simply m7 G p p p N m N A pNpNp the presence of the poly(A) tail, is required for nuclear export. As G pNpNp m7 G p p p N m N described below, the results ob5¢ cap tained in influenza-virus-infected cells have verified this conclusion. Virion RNA (b) Initiation Cellular pre-mRNAs and mRNAs contain 59 cap structures bound UpCpGpUpUpUpUpCp 10–13 nt to a complex of two proteins18,19. These cap structures and their asG p sociated proteins are not required p A m7 G p p p N m N for the nuclear export of cellular p mRNAs, but do stimulate such export. (c) Elongation Virion RNA In cells infected by influenza A virus, the nuclear export of celluUpCpGpUpUpUpUpCp lar mRNAs is blocked, and cellu7 G p p p N m N m ApGpCpApApApApGp lar pre-mRNAs and mRNAs are 1,20 degraded in the nucleus . This Viral mRNA trends in Microbiology block in nuclear export is selective: all viral mRNAs are effiFig. 1. ‘Cap-snatching’ by the influenza viral polymerase. The viral polymerase is incapable of iniciently exported21. Consequently, tiating viral mRNA synthesis unless a primer is supplied; cellular pre-mRNAs and mRNAs in the nucleus of infected cells are ‘cannibalized’ to supply these primers1. (a) An endonuclease that is viral mRNAs are the predomiintrinsic to the polymerase cleaves capped (m7 GpppNm-containing) cellular nuclear RNAs 10–13 nant newly synthesized mRNAs nucleotides (nt) from their 59 ends, preferentially at a purine residue. The resulting capped fragthat reach the translation maments serve as primers for the initiation of viral mRNA synthesis. (b) Transcription is initiated by chinery in the cytoplasm of inthe incorporation of a G residue onto the 39 end of the resulting fragments, directed by the penultimate C residue of the genome RNAs. (c) Influenza viral mRNA synthesis requires continuous fected cells, thereby contributing synthesis of cellular pre-mRNAs to provide primers for the initiation of viral mRNA chains. to the shutdown of host-cell gene expression and the selective synthesis of viral proteins. Other mechanisms must also operate to mediate this selec- to double-stranded (ds) RNA and a specific stem tive synthesis, because the cytoplasm contains many bulge in U6 small nuclear (sn) RNA31–33. NS1A exists cellular mRNAs that are synthesized prior to infec- as a dimer in vivo and in vitro, and the RNA-binding tion. Although these cellular mRNAs are stable and domain is also required for this dimerization34. The functional (as assayed by their ability to direct trans- RNA-binding and dimerization domain comprises lation in in vitro systems), their translation is selec- the amino-terminal 73 amino acids, which, in vitro, tively inhibited in infected cells by mechanisms that can function independently of the rest of the prohave not yet been elucidated20,22,23. It has been reported tein35. Nuclear magnetic resonance and X-ray crystalthat cellular mRNAs are degraded in the cytoplasm of lography have demonstrated that, in the absence of infected cells20,24,25, but this degradation occurs subse- RNA, this dimeric RNA-binding domain adopts a quent to the block in their translation20,22. novel six-helical-chain fold36–38. A second functional domain, which is located in the carboxyl half of the NS1A and the inhibition of nuclear export molecule, is not required for RNA binding, but is The NS1 protein of influenza A virus (NS1A) was nonetheless required for the inhibition of the nuclear identified in 1971 (Ref. 26), but little progress in elu- export of poly(A)-containing mRNAs29. It was postucidating its function was made until the early 1990s. lated that this domain is the effector domain that inTransient transfection experiments established that teracts with host nuclear proteins to inhibit nuclear one of its functions is the inhibition of the nuclear RNA export35, a claim which was subsequently subexport of poly(A)-containing spliced mRNAs, that stantiated. Although mutagenesis originally identified is, mRNAs without introns27–30. Nuclear export of residues 134–161 as the effector domain35, it is likely mRNAs is inhibited only when they contain a poly(A) that it extends from residue 74 (at the end of the tail produced by the cellular cleavage/polyadenyl- RNA-binding domain) to residue 237, the carboxyation system; NS1A does not inhibit the nuclear ex- terminal amino acid. port of histone mRNA, the 39 end of which is produced by a different set of factors28,30. Selective inhibition of nuclear export Mutational analysis has identified two functional CPSF and PABII domains in this 237-residue protein29. A sequence NS1A specifically inhibits the cellular 39-end processnear the amino end comprises the RNA-binding do- ing machinery by targeting two proteins: the human main, which binds with similar dissociation constants 30-kDa subunit of the cleavage and polyadenylation
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A AUA A A
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AAAAAAAAAAAAA AA A A PAP A A AAAAAAAA AAAAAA trends in Microbiology
Fig. 2. The mature 39 ends of cellular mRNAs are generated by endonucleolytic cleavage of the pre-mRNAs, followed by polyadenylation of the upstream cleavage product4,5. A minimum of two sequence elements define a poly(A) site: (1) the almost invariant AAUAAA sequence, 10–30 nucleotides upstream of the cleavage site; and (2) a variable U- or G/Urich element (DSE) located 20–40 nucleotides downstream of the cleavage site. Cleavage and poly(A) addition are carried out by a large complex of multisubunit proteins. The cleavage and polyadenylation specificity factor (CPSF) binds to the AAUAAA sequence in the pre-mRNA via its 160kDa subunit and possibly its 30-kDa subunit. Cleavage stimulation factor (CstF) binds to the DSE and stabilizes the binding of CPSF to the premRNA. Cleavage factors I and II (CFI and CFII) are also required for cleavage. Poly(A) polymerase (PAP) is also detected in the cleavage complex. After cleavage of the pre-mRNA, three factors – CstF, CFI and CFII – leave the complex, and PAP catalyses the addition of a short (~10 nucleotides) poly(A) tail. Processive elongation of the poly(A) tail requires an additional protein, poly(A)-binding protein II (PABII)43,45. PABII binds poly(A) with high affinity and specificity and, along with CPSF, tethers PAP to the RNA substrate, thereby facilitating processive synthesis of a long poly(A) tail. Cellular mRNAs containing elongated poly(A) tails are exported from the nucleus.
specificity factor (CPSF)39 and poly(A)-binding protein II (PABII)40. It is likely that these proteins bind to non-overlapping regions of the NS1A protein effector domain, and can therefore bind NS1A simultaneously40.
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These two cellular factors have different functions (Fig. 2). CPSF binds to the AAUAAA poly(A) signal located 10–30 nt upstream of the cleavage site in premRNAs and is required for both cleavage and polyadenylation of pre-mRNAs4,41. The 30-kDa subunit is one of the two CPSF subunits that have been implicated in the specific binding to the AAUAAA polyadenylation signal41,42. By contrast, PABII functions after 39 cleavage of the cellular premRNA, and is required for the processive elongation of poly(A) chains, which is catalysed by the cellular poly(A) polymerase (PAP)5,41. After PAP adds a short poly(A) tail of ~10 nt to the cleaved pre-mRNA, PABII, along with CPSF, tethers PAP to the RNA substrate, thereby facilitating processive synthesis of a long poly(A) tail in a single rapid step43. In the absence of PABII, only short poly(A) tails are added to the 39-cleaved pre-mRNA. Several lines of evidence have established that the binding of influenza virus NS1A to the 30-kDa CPSF subunit dramatically inhibits CPSF function and hence inhibits the 39-end cleavage and polyadenylation of cellular pre-mRNAs39. Immunoprecipitation experiments have shown that, in influenza-virusinfected cells, NS1A is physically associated with the 30-kDa CPSF subunit, and that this subunit is present in CPSF complexes containing all four essential protein subunits39. In vitro assays established that binding of the viral NS1A protein to the 30-kDa subunit prevents CPSF binding to the RNA substrate and inhibits 39-end cleavage and polyadenylation of cellular pre-mRNAs39. Transfection experiments demonstrated that NS1A inhibits the 39 cleavage and polyadenylation of pre-mRNAs in vivo and that the uncleaved pre-mRNAs remain in the nucleus39. Finally, influenza viruses that contain temperature-sensitive mutations in the NS1A protein-coding sequence inhibit 39 cleavage of cellular pre-mRNAs at the permissive, but not at the non-permissive, temperature44. Because NS1A inhibits the 39 cleavage of cellular pre-mRNAs, it came as a surprise that it also inhibits a reaction that occurs after cleavage, namely the processive elongation of poly(A) chains mediated by the PABII (Ref. 40). Several experiments definitively established that NS1A inhibits PABII function40. In vitro, NS1A binds PABII and inhibits its ability to stimulate the processive synthesis of long poly(A) tails catalysed by PAP. NS1A does not inhibit the binding of PABII to poly(A), and instead blocks the functional interaction of PABII with PAP. In influenza-virus-infected cells, 39 cleavage of some cellular pre-mRNAs still occurs despite the inhibition of CPSF function, followed by the addition of short poly(A) tails catalysed by the cellular PAP. These premRNAs accumulate in the nucleus of infected cells. Subsequent processive elongation of these short poly(A) tails does not occur because NS1A inhibits the function of the cellular PABII protein40. Two-pronged attack The two-pronged attack of NS1A against the cellular 39-end-processing machinery is depicted in Fig. 3.
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Cytoplasm trends in Microbiology
Fig. 3. The two-pronged mechanism by which NS1A inhibits the cellular 39-end-processing system in influenza-virus-infected cells. Pathway I: the binding of the NS1 protein to the 30-kDa subunit of CPSF blocks the binding of CPSF to the AAUAAA sequence of some cellular pre-mRNA molecules, thereby blocking 39 cleavage of these pre-mRNAs39. The uncleaved pre-mRNAs remain in the nucleus. Pathway II: CPSF binds to the AAUAAA sequence of other cellular pre-mRNA molecules, despite the binding of the NS1A protein to the 30-kDa CPSF subunit. A short poly(A) sequence is then added to these cleaved pre-mRNAs by PAP in a CPSF-dependent reaction. Subsequent elongation of the short poly(A) sequence is blocked by the binding of the NS1A protein to the PABII protein, resulting in the nuclear accumulation of cleaved pre-mRNAs containing short poly(A) tails40. In both pathways, individual NS1A protein molecules form complexes with both PABII and the 30 kDa subunit of CPSF. In these complexes the two cellular 39 processing proteins, 30 kDa CPSF and PABII, also bind directly to each other. Both species of the cellular pre-mRNAs that are sequestered in the nucleus are cleaved by the viral cap-dependent endonuclease to produce the primers required for viral mRNA synthesis. The 39 terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of 4–7 Us in the virion RNA templates. The poly(A)-containing viral mRNAs are exported from the nucleus. Reproduced, with permission, from Ref. 60. Abbreviations: CPSF, cleavage and polyadenylation specificity factor; NS1A, non-structural 1A; PAB, poly(A)-binding protein; PAP, poly(A) polymerase.
Each NS1A protein molecule in influenza-virusinfected cells forms a complex with both PABII and the 30-kDa CPSF subunit. Because it has been found that PABII and the 30-kDa CPSF subunit also bind to each other in vitro40, it is likely that all three proteins form a ternary complex. Consequently, in Fig. 3, PABII is presumed to be in the 39-processing complexes that are formed prior to 39 cleavage of the premRNA. As a result of the formation of the complex between the 30-kDa CPSF subunit and NS1A, the binding of CPSF to the AAUAAA sequence of some cellular pre-mRNA molecules is blocked, so that 39 cleavage and the subsequent addition of A residues does not occur39 (pathway I). Other cellular premRNA molecules continue to be cleaved to a certain extent in influenza-virus-infected cells, indicating that NS1A does not completely inhibit the functional binding of CPSF to the AAUAAA sequence (pathway II). It is possible that the cleavage stimulation factor (CstF), which binds to a U- or G/U-rich element
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downstream of the cleavage site4,5, might mediate formation of a 39-end-processing complex that is not effectively blocked by NS1A. Because CstF binding stabilizes the interaction of CPSF with the AAUAAA sequence4,5, the presence of a downstream sequence that affords optimal binding of CstF might be expected to offset the NS1A-mediated inhibition of CPSF binding (see Fig. 2). After the pre-mRNAs are cleaved, a short poly(A) sequence is added by PAP in a reaction that requires CPSF. Subsequent elongation of the short poly(A) sequence is blocked because NS1A inhibits PABII function40, which, along with CPSF, is required to tether PAP to poly(A) so it can catalyse the processive addition of A residues43,45. Consequently, via a two-pronged attack against PABII and CPSF, NS1A efficiently blocks the 39-end processing of cellular pre-mRNAs in influenza-virusinfected cells. Neither the uncleaved cellular pre-mRNAs (produced in pathway I) nor the cleaved cellular pre-mRNAs
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Box 1. Other viruses that regulate the nuclear export of mRNA • HIV-1: the HIV-1 Rev protein mediates the nuclear export of unspliced and partially spliced viral mRNAs, overriding their nuclear retention via spliceosome formationa. • Adenovirus: a complex of two viral proteins [E1B (55 kDa) and E4 open reading frame (ORF) 6] mediates the selective nuclear export of viral mRNAsb. • Herpes simplex virus 1: the viral ICP27 protein promotes the export of viral mRNAs, which, unlike the vast majority of cellular mRNAs, are synthesized as colinear mRNAs lacking intronsc,d. References a Cullen, B.R. (1995) Regulation of HIV gene expression. AIDS 9, S19–S32 b Dobbelstein, M. et al. (1997) Nuclear export of the E1B 55-kDa and E4 34kDa adenoviral oncoproteins mediated by a rev-like signal sequence. EMBO J. 16, 4276–4284 c Phelan, A. et al. (1996) Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts. J. Virol. 70, 5255–5265 d Sandri-Goldin, R.M. (1998) ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 6, 868–879
containing short poly(A) tails (produced in pathway II) are exported from the nucleus39,40. These observations not only confirm previous results indicating that 39-end processing of pre-mRNAs is required for their nuclear export16,17, but also indicate that nuclear export of pre-mRNAs occurs only after the PABII-mediated elongation of poly(A) chains is accomplished. By blocking the nuclear export of cellular mRNAs, the influenza viral NS1A protein inhibits the expression of cellular mRNAs synthesized after infection. In addition, the cellular pre-mRNAs and mRNAs that are trapped in the nucleus are accessible to the viral cap-dependent endonuclease for the production of the capped RNA primers that are required for viral mRNA synthesis1 (Figs 1,3). Because of the loss of their 59-cap structures, the sequestered cellular RNAs should become susceptible to nuclease digestion46 and in fact, it has been shown that cellular pre-mRNAs and mRNAs are degraded in the nucleus of infected cells20. Another mechanism The binding of the viral NS1A protein to the cellular PABII protein could also inhibit the nuclear export of cellular mRNAs by another mechanism. PABII protein molecules shuttle between the nucleus and the cytoplasm, an activity that implicates this protein in the nuclear export of cellular mRNAs40,47. The hypothesis that PABII plays an essential role in the nuclear export of cellular mRNAs is consistent with
Questions for future research • What is the mechanism by which influenza viral mRNAs are exported from the nucleus of infected cells? • Does the PABII protein participate in the nuclear export of cellular poly(A)-containing mRNAs? • What amino acid sequences of the influenza virus NS1A protein interact with the cellular 30-kDa CPSF and PABII proteins, and are similar interacting sequences found in cellular proteins?
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the finding that the 39-processing reaction, rather than simply the presence of a 39 poly(A) tail, is required for the nuclear export of cellular mRNAs16,17. It can be argued that the reason that the 39-end processing of cellular pre-mRNAs is required16,17,39,40 is that the elongated poly(A) tails bind PABII protein molecules that play an important role in mediating nuclear export. As discussed above, many other proteins that are associated with cellular pre-mRNAs and mRNAs in the nucleus have also been implicated in the nuclear export of cellular mRNAs. One possibility is that the participation of a large group of proteins in the nuclear export of mRNAs ensures that only mature, fully processed mRNAs are exported9. The presumption is that the association of these proteins with pre-mRNAs is linked to the acquisition of features of mature mRNA molecules during nuclearprocessing events. PABII would certainly link nuclear export with a processing step that is required for the production of mature mRNAs. Because NS1A inhibits the nuclear export of PABII protein molecules40, PABII would be incapable of functioning in the nuclear export of any mature cellular mRNAs that are produced despite the NS1A-mediated inhibition of 39-end processing. (See Box 1 for other viruses that regulate the nuclear export of mRNA.) Roles of the NS1A effector domain Several lines of evidence have established that the NS1A effector domain is required for the inhibition of 39-end processing of cellular pre-mRNAs. Both the 30-kDa CPSF subunit and PABII bind to the effector domain, and its deletion eliminates the ability of NS1A to inhibit 39 poly(A) processing39,40. A requirement for the RNA-binding domain, as well as the effector domain, in the inhibition of 39 poly(A) processing was suggested by early mutagenesis experiments, which indicated that mutations in the RNA-binding domain, as well as in the effector domain, result in the loss of the ability of the NS1A protein to inhibit the nuclear export of cellular mRNAs29. However, the mutant NS1A proteins used in these studies are inactive in both RNA binding and dimerization34,38. Because the loss of dimerization is a profound change in the structure of NS1A, the effector domain, as well as the RNA-binding domain, might be inactivated in such mutant proteins. To avoid this pitfall, a different mutant NS1A protein was designed based on the 3-D structure of the RNA-binding domain. This mutant lacks RNA-binding activity but retains its dimeric structure38. Because this mutant protein retains its ability to inhibit the 39-end processing of cellular premRNAs40 (Y. Li et al., submitted), it was concluded that the RNA-binding domain is not required for this inhibition, and that the effector domain alone is responsible for inhibiting 39-end processing of cellular pre-mRNAs and hence the nuclear export of cellular mRNAs. All naturally occurring influenza A viruses examined to date encode an NS1A protein that contains an effector domain48. At one time it was thought that one naturally occurring influenza A virus, A/Turkey/
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Oregon/71, encoded an NS1A protein that lacked the carboxy-terminal sequence containing the effector domain49. However, it has now been established that naturally occurring A/Turkey/Oregon/71 encodes a full-length NS1A protein48, indicating that the previously characterized virus isolate was probably generated during multiple passages in the laboratory. Consequently, the effector domain function of NS1A is conserved in nature, that is, under conditions of natural selection, indicating that this function probably plays important and necessary roles in natural infections. These necessary roles have not yet been fully delineated. The inhibition of the nuclear export of cellular mRNAs, which enhances viral gene expression, could make the virus more pathogenic and virulent in natural infections. In addition, the effector domain has one other known activity that is likely to be crucial for virus replication: an NES that is activated during infection50. However, the role of this NES in virus replication has not yet been determined. Furthermore, the effector domain might have other essential functions that have not yet been elucidated. For example, laboratory-generated viruses that lack large deletions of the effector domain fail to protect the virus against IFN (Ref. 51), indicating that the effector domain, as well as the RNA-binding domain31,32,52, might be required to protect the virus against IFN. Only in Vero cells, which are defective in IFN production, do these mutant viruses grow as well as wild-type virus in tissue culture51. The efficiency of replication of these viruses in MDCK cells, which produce IFN, is inversely correlated with the length of the effector domain of the encoded NS1A protein, and these viruses are attenuated in wild-type mice that produce IFN (Ref. 51). The requirement of the NS1A effector domain for replication in tissue culture cells has also been established by the existence of at least one mutation in the sequence of the effector domain that results in a temperature-sensitive block in virus replication44. Interestingly, although the RNA-binding domain of the NS1 protein encoded by influenza B virus (NS1B protein) possesses the same RNA-binding activities as NS1A, the NS1B effector domain does not function in the inhibition of the nuclear export of cellular mRNAs53. Consequently, this function must not be required for the propagation of influenza B viruses in nature, and the function(s) of the large carboxy-terminal region of NS1B proteins, which is encoded by all naturally occurring influenza B viruses49, is unknown. This fundamental difference between the effector domains of the NS1A and NS1B proteins probably contributes to the different biological properties of influenza A and B viruses. Conclusions and remaining issues Influenza virus NS1A blocks the nuclear export of cellular mRNAs by binding to, and inhibiting the function of, two cellular proteins that are required for the 39-end processing of cellular pre-mRNAs39,40. The inhibition of the cellular 39-end-processing machinery should not block the formation of the 39 poly(A)
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tails of viral mRNAs because the tails are produced by the viral polymerase, and not by the cellular 39-end processing machinery6,7. In fact, the influenza viral mRNAs in infected cells possess 39 poly(A) tails of 150–200 bases54. However, although the presence of elongated poly(A) tails at the 39 ends of influenza viral mRNAs is necessary for nuclear export7, it is unlikely to be sufficient. As is the case for cellular mRNAs, the nuclear export of influenza viral mRNAs is presumably mediated by one or more proteins that are bound to these mRNAs. The identity of such proteins, which could be cellular and/or virus-specific, is unknown. Because there are three different types of influenza virus mRNAs, more than one mechanism of nuclear export might operate in virus-infected cells. Of the eight influenza virus genome RNA segments, the five largest are transcribed into monocistronic mRNAs that lack introns. Only a small number of cellular genes, including histone genes, are transcribed into colinear mRNAs that lack introns55,56. It has been shown that efficient nuclear export of one of these cellular mRNAs, poly(A)-containing histone H2a mRNA, is mediated by a specific sequence in the mRNA57,58. The second type of influenza viral mRNA is encoded by the two smallest influenza virus genome segments (the M1 and NS1A mRNAs), which are transcribed into intron-containing mRNAs. Only a small proportion (~10%) of these two mRNAs is spliced, and the unspliced molecules are efficiently exported from the nucleus to be translated1. Incomplete splicing and the nuclear export of unspliced mRNAs also occurs in retrovirus-infected cells2. The nuclear export of unspliced mRNAs encoded by some retroviruses is mediated by TAP, a cellular protein that recognizes an RNA sequence in the unspliced retroviral mRNAs13,14. Like these retroviruses, influenza virus does not encode a viral protein that mediates the nuclear export of its unspliced viral mRNAs59. The viral M2 and NS2 viral mRNAs that are produced by splicing comprise the third type of influenza viral mRNA. The nuclear export of these spliced mRNAs might be similar to that of their cellular spliced mRNA counterparts, except that components of the cellular 39-end-processing system would not participate in the nuclear export of the viral M2 and NS2 mRNAs. Clearly, not much is known about the mechanism of the nuclear export of the three types of influenza viral mRNAs. Acknowledgements The work carried out in the authors’ laboratory is supported by National Institutes of Health grant AI 11772 to R.M.K. References 1 Krug, R.M. et al. (1989) Expression and replication of the influenza virus genome. In The Influenza Viruses (Krug, R.M., ed.), pp. 89–152, Plenum Press 2 O’Reilly, M.M. et al. (1995) Two strong 59 splice sites and competing, suboptimal 39 splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. Virology 213, 373–385 3 Shuman, S. (1995) Capping enzyme in eukaryotic mRNA synthesis. Prog. Nucleic Acid Res. Mol. Biol. 50, 101–129
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4 Colgan, D.F. and Manley, J.L. (1997) Mechanism and regulation of mRNA polyadenylation. Genes Dev. 11, 2755–2766 5 Wahle, E. and Kuhn, U. (1997) The mechanism of 39 cleavage and poladenylation of eukaryotic pre-mRNA. Prog. Nucleic Acid Res. Mol. Biol. 57, 41–71 6 Robertson, J.S. et al. (1981) Polyadenylation sites for influenza virus mRNA. J. Virol. 38, 157–163 7 Poon, L.L. et al. (1999) 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. 73, 3473–3476 8 Stoffler, D. et al. (1999) The nuclear pore complex: from molecular architecture to functional dynamics. Curr. Opin. Cell Biol. 11, 391–401 9 Nakielny, S. and Dreyfuss, G. (1999) Transport of proteins and RNAs in and out of the nucleus. Cell 99, 677–690 10 Pollard, V.W. et al. (1996) A novel receptor-mediated nuclear protein import pathway. Cell 86, 985–994 11 Pinol-Roma, S. and Dreyfuss, G. (1992) Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 350, 730–732 12 Nakielny, S. and Dreyfuss, G. (1996) The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals. J. Cell Biol. 134, 1365–1373 13 Pasquinelli, A.E. et al. (1997) The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16, 7500–7510 14 Gruter, P. et al. (1998) TAP, the human homolog of mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, 649–659 15 Legrain, P. and Rosbash, M. (1989) Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57, 573–583 16 Eckner, R. et al. (1991) Mature mRNA 39 end formation stimulates RNA export from the nucleus. EMBO J. 10, 3513–3522 17 Huang, Y. and Carmichael, G. (1996) Role of polyadenylation in nucleocytoplasmic transport of mRNA. Mol. Cell. Biol. 16, 1534–1542 18 Izaurralde, E. et al. (1995) A cap-binding protein complex mediating U snRNA export. Nature 376, 709–712 19 Visa, N. et al. (1996) A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J. Cell Biol. 133, 5–14 20 Katze, M.G. and Krug, R.M. (1984) Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells. Mol. Cell. Biol. 4, 2198–2206 21 Shapiro, G.I. et al. (1987) Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs. J. Virol. 61, 764–773 22 Katze, M.G. et al. (1986) Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus. J. Virol. 60, 1027–1039 23 Katze, M.G. and Krug, R.M. (1990) Translational control in influenza virus-infected cells. Enzyme 44, 265–277 24 Inglis, S.C. (1982) Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus. Mol. Cell. Biol. 1644–1648 25 Beloso, A. et al. (1992) Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff. J. Gen. Virol. 73, 575–581 26 Lazarowitz, S.G. et al. (1971) Influenza virus structural and nonstructural proteins in infected cells and their plasma membranes. Virology 46, 830–843 27 Alonso-Caplen, F.V. et al. (1992) Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev. 6, 255–267 28 Fortes, P. et al. (1994) Influenza virus NS1 protein inhibits
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pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 13, 704–712 Qian, X.Y. et al. (1994) Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA. J. Virol. 68, 2433–2441 Qiu, Y. and Krug, R.M. (1994) The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J. Virol. 68, 2425–2432 Hatada, E.R. and Fukuda, R. (1992) Binding of influenza A virus NS1 protein to dsRNA in vitro. J. Gen. Virol. 73, 3325–3329 Lu, Y. et al. (1995) Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase (PKR) that phosphorylates the eIF-2 translation initiation factor. Virology 214, 222–228 Qiu, Y. et al. (1995) The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6–U2 and U6–U4 snRNA interactions during splicing. RNA 1, 304–316 Nemeroff, M.E. et al. (1995) The influenza virus NS1 protein forms multimers in vitro and in vivo. Virology 212, 422–428 Qian, X. et al. (1995) An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA 1, 948–956 Chien, C. et al. (1997) A novel RNA-binding motif in influenza A virus non-structural protein 1. Nat. Struct. Biol. 4, 891–895 Liu, J. et al. (1997) Crystal structure of the unique multifunctional RNA-binding domain of the influenza virus NS1 protein. Nat. Struct. Biol. 4, 896–899 Wang, W. et al. (1999) RNA-binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5, 195–205 Nemeroff, M. et al. (1998) Influenza virus NS1 protein interacts with the 30kd subunit of cleavage and specificity factor and inhibits 39 end formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000 Chen, Z. et al. (1999) Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 39 end processing machinery. EMBO J. 18, 2273–2283 Barabino, S.M. and Keller, W. (1999) Last but not least: regulated poly(A) tail formation. Cell 99, 9–11 Barabino, S.M.L. et al. (1997) The 30-kd subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins. Genes Dev. 11, 1703–1716 Bienroth, S. et al. (1993) Assembly of a processive messenger RNA polyadenylation complex. EMBO J. 12, 585–594 Shimuzu, K. et al. (1999) Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology 254, 213–219 Wahle, E. and Keller, W. (1996) The biochemistry of polyadenylation. Trends Biochem. Sci. 21, 247–250 Furuichi, Y. et al. (1977) 59-terminal structure and mRNA stability. Nature 266, 235–239 Calado, A. et al. (2000) Deciphering the cellular pathway for transport of poly(A)-binding protein II. RNA 6, 245–256 Suarez, D.L. and Purdue, M.L. (1998) Multiple alignment comparison of the non-structural genes of influenza A viruses. Virus Res. 54, 59–69 Norton, G.P. et al. (1987) Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides. Virology 156, 204–213 Li, Y. et al. (1998) Regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus NS1 protein. Proc. Natl. Acad. Sci. U. S. A. 95, 4864–4869 Egorov, A. et al. (1998) Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J. Virol. 72, 6437–6441 Hatada, E. et al. (1999) Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells. J. Virol. 73, 2425–2433 Wang, W. and Krug, R.M. (1996) The RNA-binding and effector
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domains are conserved to different extents among influenza A and B viruses. Virology 223, 41–50 Plotch, S.J. and Krug, R.M. (1977) Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J. Virol. 21, 24–34 Kedes, L.H. (1979) Histone genes and histone messengers. Annu. Rev. Biochem. 48, 837–870 Nagata, S. et al. (1980) The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature 287, 401–408 Huang, Y. and Carmichael, G.G. (1997) The mouse histone H2a gene contains a small element that facilitates cytoplasmic
A well-targeted textbook Biochemistry and Molecular Biology of Antimicrobial Drug Action (5th edn) by T.J. Franklin and G.A. Snow Kluwer Academic Publishers, 1998. (£69.00/$122.13 hbk) (ix 1 166 pages) ISBN 0 412 82200 8
T
his book is essentially an updated, revised edition of the earlier fourth edition entitled Biochemistry of Antimicrobial Action. Its origins can therefore be traced to the 1970s when the first edition was published. Although there has been considerable growth in information on antimicrobial agents in the three decades since the first edition was published, the book remains admirably well focused and concise and is of comparable length to earlier editions. The broad layout of the current edition is similar to that of the fourth edition, with chapters describing the historical development of antimicrobial agents, the mechanisms of action and uptake of the principal drug classes, and the genetic and biochemical basis of resistance to antimicrobial agents. The book is written from a broad perspective as it deals with antibacterial, antifungal, antiviral and antiprotozoal agents. The principal antimycobacterial agents are also presented within the relevant
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accumulation of intronless gene transcripts and of unspliced HIV1-related mRNAs. Proc. Natl. Acad. Sci. U. S. A. 94, 10104–10109 58 Huang, Y. et al. (1999) Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. EMBO J. 18, 1642–1652 59 Alonso-Caplen, F.V. and Krug, R.M. (1991) Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rate of splicing and of the nucleocytoplasmic transport of NS1 mRNA. Mol. Cell. Biol. 11, 1092–1098 60 Lamb, R.A. and Krug, R.M. Orthomyxoviridae: the viruses and their replication. In Fields Virology (4th edn) (Knipe, D.M. and Howley, P.M., eds) Lippincott, Williams and Wilkins (in press)
target-based sections, for example, isoniazid and ethambutol are discussed in the chapter that deals with agents interfering with microbial cell wall biosynthesis. Antiseptics are also included within the definition of antimicrobial drugs. The clinical applications of the agents described are only referred to in broad terms and therefore readers wishing to gain specific information on the indications for use of a particular drug to treat disease will need to consult other texts. The previous edition of this book, somewhat confusingly for a text on antimicrobial agents, contained information on anticancer drugs; however, these descriptions have been omitted in the fifth edition, which therefore has a content more in-keeping with its title. The fifth edition now includes the phrase ‘molecular biology’ in its title to emphasize the important contributions made by molecular studies to the topic of antimicrobial agents. It is in this area that the book differs significantly from earlier editions by, for example, presenting X-ray crystallographic analysis of various bacterial blactamases and molecular descriptions of ribosome structure and multi-drug efflux systems in Gram-negative bacteria. Nevertheless, despite the comprehensive coverage of antimicrobial agents there are some important omissions. For example, neither the oxazolidinones, which represent an important new class of bacterial protein-synthesis inhibitors,
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nor the glycylcyclines (third-generation tetracycline analogues) are mentioned. Furthermore, for a book which stresses the importance of molecular biology for antimicrobial research, the significance and future impact of the extensive microbial genomesequencing projects on antimicrobial drug discovery is given only the very briefest of treatment. Despite the above criticisms, this book will be an important source of information for all those with an interest in antimicrobial agents including students, lecturers, laboratory-based molecular biologists and clinicians. However, its greatest value is likely to be as a core undergraduate textbook supporting those courses dealing with the molecular basis of antimicrobial drug action and resistance. Thus, undergraduate students will particularly benefit from the clearly written text, useful illustrations, chemical structures of drugs, and references for further reading provided at the end of each chapter. The book is also well indexed. It will certainly become a recommended text in those first and second year undergraduate modules on antibiotics that I currently teach within my own University. Ian Chopra Divn of Microbiology and Antimicrobial Research Centre, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK LS2 9JT
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