Journal of Antimicrobial Chemotherapy (1975) 1 {SuppQ, 9-17
The replication of influenza Tiros RNA
Richard D . Barry
Introduction
The incidence of virus diseases has not been altered appreciably by chemotherapy, despite extensive screening programmes carried out over several decades. This failure has been attributed to the special features of viruses. Extracellular virus particles are metabolically inert and their multiplication has an obligatory dependence on cellular metabolic processes. Once inside cells, viruses stimulate a rapid increase in the rate of synthesis of certain classes of macromolecule, particularly nucleic acids and proteins, so that essentially normal activities of cells provide the components from which progeny virus particles are assembled. Although it has been known for many years that certain stages of virus replication cycle, such as uptake, uncoating or assembly provide targets for chemotherapeutic attack, success so far has been very limited, even allowing for the effectiveness of such compounds as Amantadine in inhibitingthe process of "penetration" by influenza viruses. Recent developments in our understanding of the biochemistry of the virus replication have brought to light novel aspects of the multiplication process, particularly in regard to the synthesis and function of nucleic acids and this detailed knowledge should greatly improve the prospects for developing compounds that may act as specific inhibitors of virus replication. The purpose of this paper is to consider certain features of the replication of influenza viruses concentrating particularly on the means by which the genetic constituents of the virus (the genome) function and are reproduced. Influenza viruses are irregularly spherical, with a lipid containing outer membrane in which are located the two distinct glycoprotein structural entities, the haemagglutinin and neuraminidase. The outer membrane encloses a structural shell, consisting entirely of the M or matrix protein. Located internally is the ribonucleoprotein (RNP), containing the ribonucleic acid (RNA) genome of the virus. Influenza viruses are still popularly known as myxoviruses, although it is now the convention to classify them as 'orfAomyxoviruses', so as to distinguish them from viruses of the ^ara-influenza type that are now called 'paramyxoviruses' (Andrewes & Pereira, 1972). Knowledge of the general principles of RNA virus replication has accumulated rapidly in recent years. The finding that small RNA-containing viruses multiply normally in the absence of DNA synthesis (Simon, 1961; Cooper & Zinder, 1962) or DNA-directed RNA synthesis (Reich, Franklin, Shatkin & Tatum, 1961)firmlyestablished the concept
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, England
10
R. D. Barry
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
that viral RNA replication is independent of the genetic material of the host cell. The detection of ribonuclease-resistant, virus-specific RNA in cells infected with EMC virus (Montagnier & Saunders, 1963) implied that viral RNA replicates by way of a double stranded, base-paired intermediate. The existence of such replicative forms has been established for many animal and bacterial RNA viruses (Bishop & Levintow, 1971) and it is now accepted that the replication of viral RNA is a 2-stage process, in which the first step is the synthesis of a complementary polynucleotide strand that acts as a template in the second step for the synthesis of progeny strands of viral RNA; in the case of the RNA tumour viruses, the complementary polynucleotide template is DNA (Temin, 1970). In those instances where the isolated viral RNA is infectious, the RNA serves as both genome and messenger RNA (plus strand), while the complementary RNA (minus strand) acts only as a template for plus strand formation. Cells infected with RNA viruses contain virus-specific RNA-dependent RNA polymerase(s) (Baltimore & Franklin, 1962; Baltimore, Eggers, Franklin & Tamm, 1963); these enzymes are not found in uninfected cells and they are responsible for the synthesis of both template and progeny RNA. The enzyme specifically concerned with progeny RNA synthesis is referred to as a replicase. The RNA of the orthomyxoviruses lacks infectivity and the mRNA associated with polysomes of cells infected by these viruses is complementary to virion RNA (Pons, 1972). Hence, influenza viruses differ from most other RNA viruses in that vRNA is not mRNA. Together with the paramyxoviruses and the rhabdoviruses, they have been described collectively as "Negative Strand Viruses" (Mahy & Barry, 1975). An essential requirement for this type of virus is the presence within the virion of an RNA polymerase capable of transcribing the virion RNA into mRNA following infection. Such a virus associated transcriptase has been found in the influenza viruses (Chow & Simpson, 1971; Penhoet, Miller, Doyle & Blatti, 1971). Certain general features of the multiplication of influenza viruses can be stated. These viruses contain single stranded, non-infectious RNA that occurs in segments (Davies & Barry, 1966). The total molecular weight of the RNA of each particle has been estimated to be 5 X 10s daltons, made up of 8-9 pieces (Skehel, 1972). Influenza viruses have a transcriptase that catalyses the formation of cRNA in vitro (Bishop, Obijeski & Simpson, 1971) and the reaction is not affected by the presence of actinomycin D (Chow & Simpson, 1971). When cells are infected in the presence of actinomycin D however, virus growth is inhibited (Barry, Ives & Cruickshank, 1962) and no transcription occurs (Bean & Simpson, 1973). Influenza viruses cannot replicate in anucleate cell fragments (Cheyne & White, 1969), enucleated cells (Follett, Pringle, Wunner & Skehel, 1974) or in cells in which there has been an inhibition of DNA function (Barry, 1964) or DNA transcription (Mahy, Hastie & Armstrong, 1972). This evidence suggests that, unlike any other RNA virus, the replication of the orthomyxoviruses may require the participation of the genetic material of their host cells. The following events concerned with RNA synthesis in cells infected by influenza viruses occur. There is an initial period following infection of approximately 40 min duration when virus-induced RNA synthesis cannot be detected. This period of maximum inhibitor sensitivity is followed by the appearance and rapid accumulation of complementary (c) RNA (Bean & Simpson, 1973). A significant proportion of cRNA rapidly becomes associated with the polyribosomes (Glass, McGeoch & Barry, 1975) to provide mRNA for the production of virus specific polypeptides. The synthesis of cRNA reaches a peak at 2-2| h after infection (Glass et al., 1975) whereas the synthesis of progeny
Influenza virus RNA
11
virion (v) RNA begins later than that of cRNA, beginning approximately 90 min after infection and reaching a maximum at 3 h (Scholtissek, Rott, Hansen, Hansen & Schafer, 1962). The formation and release of newly assembled virus begins about 4 h following infection. Various aspects of influenza virus-induced RNA synthesis will now be considered in more detail. Transcription
Table L Requirements for in vitro RNA polymerase activity of influenza virus (FPV) particles
Complete* —ATP, GTP & CTP -Mn1 + -Mg' + - M n 1 + andMg i + -NP40 —Dithiothreitol + C a t + 1 mM +RNase A 100 ng/ml +Actinomycin D 200 ng/ml +Rifampicin 200 ng/ml +Streptolydigin 200 ng/ml +Rifamcyin AF/013 150 jig/ml
100 2 34 42 0 0 60 2 0 100 100 100 100
• The complete assay mixture contained the following components: 50 mM Tris HC1 buffer (pH 8-2), 2 0 mM each of ATP, CTP and GTP, 0-4 mM [3H]UTP (32 Ci/mol), 8 mM MgCl,, 0-2mMMnCl,, 5 mM-Dithiothreitol, 150 mM KCI, 33 mM NaCl and 0-5% Nonidet-P40. The total reaction volume was 150 nl containing 50-150 ng of viral protein. Reaction mixtures were incubated at 31°C and duplicate samples of 50 id 1 were removed and spotted on the Whatman GF-A glass fibre niters and dried at 60°C for 2 min. The filters were then washed in 0-5 N TCA containing 0 1 M sodium pyrophosphate ( x 1), 0-5 N TCA ( x 4 ) and finally in ethanol and then dried at 60°C. When dry the filters were counted in a liquid scintillation counter in a toluene-based scintillant. Background counts were subtracted and specific activities calculated. A typical 100% value was 3 nmol/mg of protein/h.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
The virion transcriptase RNA-dependent transcriptases have been identified in both influenza A viruses (Chow & Simpson, 1971; Penhoet et al., 1971; Skehel, 1971) and influenza B viruses (Oxford, 1973). Transcriptase activity can be detected by the incorporation of a radioactively labelled ribonucleoside triphosphate into acid-insoluble product when the reaction mixture contains virus particles, detergents such as Triton N 101 or Nonidet P40, divalent cations and all 4 ribonucleoside triphosphates. The complete ingredients of a typical reaction mixture are listed in the footnote to Table I, and the following features can be noted. The reaction is dependent on the presence of all 4 ribonucleoside triphosphates and only proceeds when the virus particles are disrupted by detergent, implying that the enzyme is located inside the virus particle. The template for the reaction is RNA, since incorporation is completely blocked by the presence of ribonuclease. Deoxyribonuclease
12
R.D. Barry
Transcription in vivo The obvious role for virion-associated transcriptase is to provide cRNA that is needed both as a template for the production of progeny vRNA and as mRNA for polypeptide synthesis. The term primary transcription (Flamand & Bishop, 1973) has been coined to describe RNA synthesis due to the incoming virion polymerase molecules can can be observed in infected cells treated with protein synthesis inhibitors such as cycloheximide (Bean and Simpson, 1973). An unusual feature of primary transcription by influenza viruses is that a delay of 40 min takes place before it begins, and the process is inhibited completely by the presence of actinomycin D (Bean & Simpson, 1973). These findings suggest that during the initial delay period, some host cell activity occurs that controls the synthesis of cRNA by the transcriptase. These cell functions are presumably mediated by newly synthesized nucleic acids rather than proteins and their nature is presently inexplicable. Primary transcription continues at a relatively low level until 90 min after infection, after which time it accelerates rapidly. This amplification is apparently brought about by the synthesis of new transcriptase molecules, because it does not occur in cells treated with protein synthesis inhibitors.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
has no effect on the reaction (Chow & Simpson, 1971). Inhibitors of DNA-dependent polymerases, such as actinomycin D, do not affect the reaction. Maximum activity for the reaction depends on the presence of both Mn w and Mg*+ ions (Carroll, McGeoch & Many, 1975). The levels of enzyme activity associated with different strains of influenza virus were found to vary over an approximately 20-fold range (Chow & Simpson, 1971). However, these differences disappear when reaction mixtures are incubated with 0-3 mM G P G (see below). This suggests that the observed strain differences are due to differences in the initiation of RNA synthesis in vitro, rather than quantitative difference in levels of enzyme between different strains (Carroll et al., 1975). Under optimal conditions, at least 80 % of the genome is transcribed in vitro (Bishop, Roy, Bean & Simpson, 1972; Carroll et al, 1975) but the process is slow and nonrepetitive. The product of the reaction appears to be entirely complementary to vRNA, suggesting that the primary function of this virus-associated enzyme is to synthesize mRNA; replicase activity has not been ascribed to this enzyme. Hefti, Roy & Bishop (1975) found that, although there are multiple initiation sites for the influenza virus transcriptase, the initial sequence for each site is unique, beginning pppGpCp. The activity of the virion-associated transcriptase is stimulated by guanosine and, depending on the strain of virus used, the stimulation of the initial reaction is up to 10-fold (McGeoch & Kitron, 1975). 5'-GMP, 3',5'-cyclic GMP and 5'-GDP show lesser stimulation effects. No other nucleosides or 5'-NMPs have any, but the dinucleoside monophosphates GpG and GpC show large stimulations. Stimulation represents preferential initiation of cRNA synthesis and guanosine is incorporated specifically at the 5' terminus of the progeny RNA. This is the first demonstration of initiation of synthesis of a nucleic acid chain with a nucleoside and suggests that initiation of RNA synthesis by transcriptase might be a suitable target for chemotherapy. The activation effects of GpG and GpC suggest that they too act as chain initiators. It is not known for certain what structural polypeptides constitute the transcriptase, as the enzyme is bound tightly to the nucleocapsid (RNP) structures, but three minor components of the virus designated as polypeptides P^ P 2 and P8, ranging in molecular weight from 85 000-96 000 are always associated with the transcription complex and seen likely candidates as structural components of the enzyme.
Influenza virus RNA
13
Influenza virus messenger RNA
Replication The role of the cell nucleus Any discussion of the replication of influenza virus RNA would be incomplete without a consideration of the role played by cell nuclei in this process. Influenza viruses require the presence of a cell nucleus before any virus-directed RNA or protein synthesis can take place (Follett et al., 1974; Kelly, Avery & Dimmock, 1974). Since these viruses do not multiply in the presence of inhibitors of DNA function such as actinomycin D or mitomycin C (Barry et al, 1962; Barry, 1964; Rott, Saber & Scholtissek, 1965; Nayak & Rasmussen, 1966) or in the presence of a-amanitin (Rott & Scholtissek, 1970; Mahy et al., 1972) it is likely that cellular DNA-dependent RNA synthesis is required for the initiation of successful infection. The possibility that an individual component of the segmented RNA genome of the virus is a cell gene product has been excluded by detailed hybridization studies (Cox & Barry, 1975). The functional significance of cell gene transcription in influenza replication is not yet understood.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
RNA complementary to vRNA has been detected in polyribosome fractions of infected cells and has properties expected of mRNA (Pons, 1972; Etkind & Krug, 1974). In a detailed characterization of influenza mRNA associated with polyribosomes Glass et al. (1975) found that virus mRNA was first detected at 45 min after infection. As the synthesis of total cRNA is just beginning at this time (Bean & Simpson, 1973), nascent cRNA must be rapidly processed into polyribosomal RNA. Virus mRNA was found both on membrane bound and free polyribosomes, it contained poly(A), and reached its maximum rate of appearance at 2-2-5 h after infection. From 45 min onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80 % of the genome RNA, while poly(A)-minus RNA contained sequences complementary to 90-100% of the genome. It has been reported (Skehel, 1973) that some virus proteins are synthesized in detectable amounts earlier in infection than others, implying that part of the genome RNA may be transcribed at an earlier stage of infection than the rest. Data to support this idea have been provided by Avery & Dimmock (1975). In contrast, Glass et al. (1975) found that even at the earliest stages of infection mRNA sequences complementary to more than 90% of the genome RNA were being synthesized. They found, however, that early in infection there was a gross asymmetry in sequence representation, in that almost half the genome appeared to be represented in much greater quantity than the other half. From this they concluded that influenza viruses do not exhibit temporal control of transcription. It has been demonstrated recently that many eukaryotic and viral mRNAs undergo modification of their 5'-termini by methylation, an apparent prerequisite for translation (Shatkin, Banerjee, Both, Furuichi & Nuthukrishnan, 1975). The modified structure at the 5'-terminus is an inverted nucleotide, 7-methylguanylic acid (m'G) and its addition to the mRNA is effected by pyrophosphate linkage and methylation at the 2'-position of the first nucleotide at the 5'-terminus of mRNA in the presence of the methyl-donor S-adenosylmethionine (SAM) (Miura et al., 1975). This process is known as "blocking" or "capping" and has been demonstrated in vitro when the influenza transcriptase product is synthesized in the presence of SAM (Miura et al., 1975). The implication is that influenza mRNA is terminally modified in vivo.
14
R. D. Barry
Virus-specific RNA replication RNA with the characteristics of a base-paired replicative form (Duesberg & Robinson, 1967) and replicative intermediate (Nayak, 1970) have been identified in infected cells. As mentioned above, the formation of cRNA preceeds that of vRNA; synthesis of vRNA reachs a peak at 3 h after infection (Scholtissek & Rott, 1970) and is the predominant RNA synthesized from 4 h onwards (Krug, 1972). The replicase enzyme responsible for the synthesis of vRNA from the cRNA template has not yet been identified, despite an intensive search. Ho & Walters (1966) detected an RNA-dependent RNA polymerase in the microsomal fraction of infected cells and for some time it was assumed that this enzyme was responsible for vRNA synthesis. More recently, an RNA-dependent RNA polymerase was found in highly purified nuclei from influenza-infected cells and reached maximum activity at 3-4 h (Hastie & Mahy, 1973). However, not only are the characteristics of both nuclear and cytoplasmic enzymes very similar, but the product synthesized by both is predominantly cRNA, i.e. both enzymes seem to have the properties of a transcriptase rather than a replicase. At present the nature of the replicase remains obscure, but at least 3 possibilities are worth consideration: (1) that replication and transcription are carried out by the same enzyme; (2) that a presently unrecognized, virus-induced protein acts as the replicase; or (3) that the transcription enzyme is modified by the addition of an extra protein, to form the replicase. If replication and transcription are different aspects of the activity of a single structural entity, it is surprising that the in vitro product of the variously isolated RNA polymerases is invariably cRNA. It may be that conditions in vitro are far from ideal, in that replication is never initiated, or the cRNA may require modification, such as the acquisition of a protein coat, before it can act as a replication intermediate. The alternative possibility, namely that another protein, either singly or in combination with the existing transcriptase, acts as the replicase, seems more likely. Of the virusinduced proteins found in cells infected by influenza virus the most likely candidate would be the non-structural (NS) polypeptide, to which a function has not yet been ascribed. In conclusion, several aspects of RNA synthesis in cells infected by influenza viruses are not yet understood. Cell nuclear activities obviously play an important role in the initiation of RNA synthesis. It is not known why cRNA synthesis predominates early in
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Apart from the need for cell gene transcription, it is likely that events associated with virus-induced RNA synthesis take place in the cell nucleus. It is known for instance that at least 2 virus-induced proteins, the RNP and a non-structural protein, accumulate in infected cell nuclei (Breitenfeld & Schafer, 1957; Dimmock & Watson, 1969). An early report (Scholtissek et al., 1962) suggested that virus particle-associated RNA was made in the nucleus. However, cell fractionation studies revealed the presence of newly synthesized virus RNA in both the cytoplasm and nucleoplasm, although cRNA was predominately cytoplasmic (Krug, 1972). The topographical studies based on autoradiography (Armstrong & Barry, 1974) suggest that major phases of virus-induced RNA synthesis occur in cell nuclei. Nuclei may be important in influenza virus replication for at least 3 reasons. In the first place, RNA synthesis directed by host DNA is needed in the early stages of replication. Following this, nuclei may provide not only the site of viral RNA synthesis, but also the site of assembly of the RNP.
Influenza virus RNA
15
infection, or what factors are responsible for the switch from cRNA to vRNA synthesis. Furthermore, the structural components of either the transcriptase orreplicasehave not been identified directly and it is not known whether either of them require host factors for in vivo activity. The overall interrelationships of viral replication to RNA transcription are a virgin field of study, with important questions yet to be asked concerning host specificity and viral to host interrelationships (Bishop & Flamand, 1975). References
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Andrewes, C. H. & Pereira, H. G. Viruses of Vertebrates. Bailliere Tindall, London (1972), 3rd edition. Armstrong, S. J. & Barry, R. D. The topography of RNA synthesis in cells infected with fowl plague virus. Journal of General Virology 24: 535-47 (1974). Avery, R. J. & Dimmock, N. J. Temporal control of transcription of influenza virus RNA. Virology 64: 409-14 (1975). Baltimore, D. & Franklin, R. M. Preliminary data on a virus specific enzyme system responsible for the synthesis of viral RNA. Biochemical and Biophysical Research Communications 9: 388-92 (1961). Baltimore, D., Eggers, H. J., Franklin, R. M. & Tamm, I. Poliovirus induced RNA polymerase and the effects of virus specific inhibitors on its production. Proceedings of the National Academy of Sciences of the United States of America 49: 843-9 (1963). Barry, R. D. The effects of actinomycin D and ultraviolet irradiation on the formation of fowl plague virus. Virology 24: 398-405 (1964). Barry, R. D., Ives, D. R. & Cruickshank, J. G. Participation of deoxyribonucleic acid in the multiplication of influenza virus. Nature 194: 1139-40 (1962). Bean, W. J. & Simpson, R. W. Primary transcription of the influenza virus genome in permissive cells. Virology 56: 646-51 (1973). Bishop, D. H. L. & Flamand, A. Transcription processes of animal RNA viruses. Control Processes in Virus Multiplication, Society for General Microbiology, 25th Symposium, Cambridge University Press (1975), pp. 95-152. Bishop, J. M. & Levintow, L. Replicative forms of viral RNA. Progress in Medical Virology 13: 1-82(1971). Bishop, D. H. L., Obijeski, J. F. & Simpson, R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. 1. Optimal conditions for in vitro activity of the ribonucleic acid dependant ribonucleic acid polymerase. Journal of Virology %: 66-73 (1971). Bishop, D. H. L., Roy, P., Bean, W. J. & Simpson, R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. Ill Completeness of the transcription process. Journal of Virology 10: 689-97 (1972). Breitenfeld, P. M. & Schafer, W. The formation of fowl plague virus antigens in infected cells as studied with fluorescent antibodies. Virology 4: 328-45 (1957). Carroll, A. R., McGeoch, D. & Mahy, B. W. J. In vitro transcription by the influenza virion RNA transcriptase. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47: 95-102 (1975). Cheyne, I. M. & White, D. O. Growth of paramyxoviruses in anucleate cells. Australian Journal of Experimental Biology and Medical Science 47: 145-7 (1969). Chow, N-L. & Simpson, R. W. RNA dependent RNA polymerase activity associated with virions and sub-viral particles of myxovirus. Proceedings of the National Academy of Science of the United States of America 68: 752-6 (1971). Cooper, S. & Zinder, N. D. The growth of RNA bacteriophage: role of DNA synthesis. Virology 18: 405-11 (1962). Cox, N. J. & Barry, R. D. Hybridization studies of the relationship between influenza virus RNA and cellular DNA. Virology (1975, in press). Davies, P. & Barry, R. D. Nucleic acid of influenza virus. Nature 211: 384-7 (1966). Dimmock, N. J. & Watson, D. H. Proteins specified by influenza virus in infected cells: analysis by polyacrylamide gel electrophoresis of antigens not present in the virus particle. Journal of General Virology 5: 499-509 (1969).
16
R. D. Barry
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Duesberg, P. H. & Robinson, W. S. On the structure and replication of influenza virus. Journal of Molecular Biology 25: 383-405 (1967). Etkind, P. R. & Krug, R. M. Influenza viral messenger RNA. Virology 62: 38-45 (1974). Flamand, A. & Bishop, D. H. L. Primary in vivo transcription of vesicular stomatitis virus and temperature-sensitive mutants of five vesicular stomatitis virus complementation groups. Journal of Virology 12: 1238-52 (1973). Follett, E. A. C , Pringle, C. R., Wunner, W. H. & Skehel, J. J. Virus replication in enucleate cells: vesicular stomatitis virus and influenza virus. Journal of Virology 13: 394-9 (1974). Glass, S. E., McGeoch, D. & Barry, R. D. Characteristics of the messenger RNA of influenza virus. Journal of Virology (1975, in press). Hastie, N. D. & Mahy, B. W. J. RNA polymerase in nuclei of cells infected with influenza virus. Journal of Virology 12: 951-61 (1973). Hefti, E., Roy, P. & Bishop, D. H. L. The initiation of transcription by influenza virion transcription. Negative Strand Viruses. Academic Press, London (1975), pp. 307-26. Ho, P. P. K. & Walters, P. Influenza virus induced ribonucleic acid nucleotidyl transferase and the effect of actinomycin D on its formation. Biochemistry 5: 231-5 (1966). Kelly, D. C , Avery, R. J. & Dimmock, N. J. Failure of an influenza virus to initiate infection in enucleate BHK cells. Journal of Virology 13: 1155-61 (1974). Krug, R. M. Cytoplasmic and nucleoplasmk viral RNPs in influenza-virus-infected MDCK cells. Virology 50: 103-13(1972). McGeoch, D. & Kitron, N. Influenza virion RNA dependent RNA polymerase: stimulation by guanosine and related components. Journal of Virology 15: 686-95 (1975). Mahy, B. W. J. & Barry, R. D. Negative Strand Viruses. Academic Press, London (1975). Mahy, B. W. J., Hastie, N. D. & Armstrong, S. J. Inhibition of influenza virus replication by a-amanitin: mode of action. Proceedings of the National Academy of Sciences of the United States of America 69: 1421-4 (1972). Montagnier, L. &Saunders, F. K. Replication forms of encephelomyocarditis virus ribonucleic acid. Nature 199: 664-7 (1963). Miura, K., Furuichi, Y., Shimotohno, K., Urushibara, T., Watanabe, K. & Suguira M. Modification at the 5'-terminus of messenger RNA strand. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47: 153-60 (1975). Nayak, D. P. Replication of influenza virus RNA. Biology ofLarge RNA viruses. Academic Press, London (1970), pp. 371-91. Nayak, D. P. & Rasmussen, A. F. J. Influence of mitomycin C on the replication of influenza viruses. Virology 30: 673-83 (1966). Oxford, J. S. Polypeptide composition of influenza B viruses and enzymes associated with the purified virus particles. Journal of Virology 12: 827-35 (1973). Penhoet, E., Miller, H., Doyle, M. & Blatti, S. RNA dependent RNA polymerase activity in influenza viruses. Proceedings of the National Academy of Sciences of the United States of America 68: 1369-71 (1971). Pons, M. Studies on the replication of influenza virus RNA. Virology 47: 823-32 (1972). Reich, E., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. Effect of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134: 556-7 (1961). Rott, R., Saber, S. & Scholtissek, C. Effect on myxoviruses of mitomycin C, actinomycin D and pretreatment of the host cell with ultraviolet light. Nature 205: 1187-90 (1965). Rott, R. & Schottissek, C. Specific inhibition of influenza replication by a-amanitin. Nature 228: 56 (1970). Schottissek, C. & Rott, R. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40: 989-96 (1970). Schottissek, C , Rott, R., Hausen, P., Hausen, H. & Schafer, W. Comparative studies of RNA and protein synthesis with a myxovirus and a small polyhedral virus' Cold Spring Harbor Symposium of Quantitative Biology 27: 245-57 (1962). Shatkin, A. J., Baneriee, A. K., Both, G. W., Furuichi, Y. & Nuthukrishnan, S. 5'-terminal 7-methylguanosine in eukaryotic mRNAs and its requirement for translation. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47:177-36 (1975). Skehel, J. J. RNA-dependent polymerase activity of the influenza virus. Virology 45: 793-6 (1971).
Influenza virus RNA
17
Skehel, J. J. Polypeptide synthesis in influenza vims infected cells. Virology 49: 23-6 (1972). Skehel, J. J. Early polypeptide synthesis in influenza virus infected cells. Virology 56: 394-9 (1973). Simon, E. H. Evidence for the non-participation of DNA in viral RNA synthesis. Virology 13: 105-18 (1961). Temin, H. M. Formation and activation of the provirus of RNA sarcoma viruses. Biology of Large RNA viruses. Academic Press, London (1970), pp. 233-49.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
The replication of influenza Tiros RNA
Richard D . Barry
Introduction
The incidence of virus diseases has not been altered appreciably by chemotherapy, despite extensive screening programmes carried out over several decades. This failure has been attributed to the special features of viruses. Extracellular virus particles are metabolically inert and their multiplication has an obligatory dependence on cellular metabolic processes. Once inside cells, viruses stimulate a rapid increase in the rate of synthesis of certain classes of macromolecule, particularly nucleic acids and proteins, so that essentially normal activities of cells provide the components from which progeny virus particles are assembled. Although it has been known for many years that certain stages of virus replication cycle, such as uptake, uncoating or assembly provide targets for chemotherapeutic attack, success so far has been very limited, even allowing for the effectiveness of such compounds as Amantadine in inhibitingthe process of "penetration" by influenza viruses. Recent developments in our understanding of the biochemistry of the virus replication have brought to light novel aspects of the multiplication process, particularly in regard to the synthesis and function of nucleic acids and this detailed knowledge should greatly improve the prospects for developing compounds that may act as specific inhibitors of virus replication. The purpose of this paper is to consider certain features of the replication of influenza viruses concentrating particularly on the means by which the genetic constituents of the virus (the genome) function and are reproduced. Influenza viruses are irregularly spherical, with a lipid containing outer membrane in which are located the two distinct glycoprotein structural entities, the haemagglutinin and neuraminidase. The outer membrane encloses a structural shell, consisting entirely of the M or matrix protein. Located internally is the ribonucleoprotein (RNP), containing the ribonucleic acid (RNA) genome of the virus. Influenza viruses are still popularly known as myxoviruses, although it is now the convention to classify them as 'orfAomyxoviruses', so as to distinguish them from viruses of the ^ara-influenza type that are now called 'paramyxoviruses' (Andrewes & Pereira, 1972). Knowledge of the general principles of RNA virus replication has accumulated rapidly in recent years. The finding that small RNA-containing viruses multiply normally in the absence of DNA synthesis (Simon, 1961; Cooper & Zinder, 1962) or DNA-directed RNA synthesis (Reich, Franklin, Shatkin & Tatum, 1961)firmlyestablished the concept
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, England
10
R. D. Barry
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
that viral RNA replication is independent of the genetic material of the host cell. The detection of ribonuclease-resistant, virus-specific RNA in cells infected with EMC virus (Montagnier & Saunders, 1963) implied that viral RNA replicates by way of a double stranded, base-paired intermediate. The existence of such replicative forms has been established for many animal and bacterial RNA viruses (Bishop & Levintow, 1971) and it is now accepted that the replication of viral RNA is a 2-stage process, in which the first step is the synthesis of a complementary polynucleotide strand that acts as a template in the second step for the synthesis of progeny strands of viral RNA; in the case of the RNA tumour viruses, the complementary polynucleotide template is DNA (Temin, 1970). In those instances where the isolated viral RNA is infectious, the RNA serves as both genome and messenger RNA (plus strand), while the complementary RNA (minus strand) acts only as a template for plus strand formation. Cells infected with RNA viruses contain virus-specific RNA-dependent RNA polymerase(s) (Baltimore & Franklin, 1962; Baltimore, Eggers, Franklin & Tamm, 1963); these enzymes are not found in uninfected cells and they are responsible for the synthesis of both template and progeny RNA. The enzyme specifically concerned with progeny RNA synthesis is referred to as a replicase. The RNA of the orthomyxoviruses lacks infectivity and the mRNA associated with polysomes of cells infected by these viruses is complementary to virion RNA (Pons, 1972). Hence, influenza viruses differ from most other RNA viruses in that vRNA is not mRNA. Together with the paramyxoviruses and the rhabdoviruses, they have been described collectively as "Negative Strand Viruses" (Mahy & Barry, 1975). An essential requirement for this type of virus is the presence within the virion of an RNA polymerase capable of transcribing the virion RNA into mRNA following infection. Such a virus associated transcriptase has been found in the influenza viruses (Chow & Simpson, 1971; Penhoet, Miller, Doyle & Blatti, 1971). Certain general features of the multiplication of influenza viruses can be stated. These viruses contain single stranded, non-infectious RNA that occurs in segments (Davies & Barry, 1966). The total molecular weight of the RNA of each particle has been estimated to be 5 X 10s daltons, made up of 8-9 pieces (Skehel, 1972). Influenza viruses have a transcriptase that catalyses the formation of cRNA in vitro (Bishop, Obijeski & Simpson, 1971) and the reaction is not affected by the presence of actinomycin D (Chow & Simpson, 1971). When cells are infected in the presence of actinomycin D however, virus growth is inhibited (Barry, Ives & Cruickshank, 1962) and no transcription occurs (Bean & Simpson, 1973). Influenza viruses cannot replicate in anucleate cell fragments (Cheyne & White, 1969), enucleated cells (Follett, Pringle, Wunner & Skehel, 1974) or in cells in which there has been an inhibition of DNA function (Barry, 1964) or DNA transcription (Mahy, Hastie & Armstrong, 1972). This evidence suggests that, unlike any other RNA virus, the replication of the orthomyxoviruses may require the participation of the genetic material of their host cells. The following events concerned with RNA synthesis in cells infected by influenza viruses occur. There is an initial period following infection of approximately 40 min duration when virus-induced RNA synthesis cannot be detected. This period of maximum inhibitor sensitivity is followed by the appearance and rapid accumulation of complementary (c) RNA (Bean & Simpson, 1973). A significant proportion of cRNA rapidly becomes associated with the polyribosomes (Glass, McGeoch & Barry, 1975) to provide mRNA for the production of virus specific polypeptides. The synthesis of cRNA reaches a peak at 2-2| h after infection (Glass et al., 1975) whereas the synthesis of progeny
Influenza virus RNA
11
virion (v) RNA begins later than that of cRNA, beginning approximately 90 min after infection and reaching a maximum at 3 h (Scholtissek, Rott, Hansen, Hansen & Schafer, 1962). The formation and release of newly assembled virus begins about 4 h following infection. Various aspects of influenza virus-induced RNA synthesis will now be considered in more detail. Transcription
Table L Requirements for in vitro RNA polymerase activity of influenza virus (FPV) particles
Complete* —ATP, GTP & CTP -Mn1 + -Mg' + - M n 1 + andMg i + -NP40 —Dithiothreitol + C a t + 1 mM +RNase A 100 ng/ml +Actinomycin D 200 ng/ml +Rifampicin 200 ng/ml +Streptolydigin 200 ng/ml +Rifamcyin AF/013 150 jig/ml
100 2 34 42 0 0 60 2 0 100 100 100 100
• The complete assay mixture contained the following components: 50 mM Tris HC1 buffer (pH 8-2), 2 0 mM each of ATP, CTP and GTP, 0-4 mM [3H]UTP (32 Ci/mol), 8 mM MgCl,, 0-2mMMnCl,, 5 mM-Dithiothreitol, 150 mM KCI, 33 mM NaCl and 0-5% Nonidet-P40. The total reaction volume was 150 nl containing 50-150 ng of viral protein. Reaction mixtures were incubated at 31°C and duplicate samples of 50 id 1 were removed and spotted on the Whatman GF-A glass fibre niters and dried at 60°C for 2 min. The filters were then washed in 0-5 N TCA containing 0 1 M sodium pyrophosphate ( x 1), 0-5 N TCA ( x 4 ) and finally in ethanol and then dried at 60°C. When dry the filters were counted in a liquid scintillation counter in a toluene-based scintillant. Background counts were subtracted and specific activities calculated. A typical 100% value was 3 nmol/mg of protein/h.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
The virion transcriptase RNA-dependent transcriptases have been identified in both influenza A viruses (Chow & Simpson, 1971; Penhoet et al., 1971; Skehel, 1971) and influenza B viruses (Oxford, 1973). Transcriptase activity can be detected by the incorporation of a radioactively labelled ribonucleoside triphosphate into acid-insoluble product when the reaction mixture contains virus particles, detergents such as Triton N 101 or Nonidet P40, divalent cations and all 4 ribonucleoside triphosphates. The complete ingredients of a typical reaction mixture are listed in the footnote to Table I, and the following features can be noted. The reaction is dependent on the presence of all 4 ribonucleoside triphosphates and only proceeds when the virus particles are disrupted by detergent, implying that the enzyme is located inside the virus particle. The template for the reaction is RNA, since incorporation is completely blocked by the presence of ribonuclease. Deoxyribonuclease
12
R.D. Barry
Transcription in vivo The obvious role for virion-associated transcriptase is to provide cRNA that is needed both as a template for the production of progeny vRNA and as mRNA for polypeptide synthesis. The term primary transcription (Flamand & Bishop, 1973) has been coined to describe RNA synthesis due to the incoming virion polymerase molecules can can be observed in infected cells treated with protein synthesis inhibitors such as cycloheximide (Bean and Simpson, 1973). An unusual feature of primary transcription by influenza viruses is that a delay of 40 min takes place before it begins, and the process is inhibited completely by the presence of actinomycin D (Bean & Simpson, 1973). These findings suggest that during the initial delay period, some host cell activity occurs that controls the synthesis of cRNA by the transcriptase. These cell functions are presumably mediated by newly synthesized nucleic acids rather than proteins and their nature is presently inexplicable. Primary transcription continues at a relatively low level until 90 min after infection, after which time it accelerates rapidly. This amplification is apparently brought about by the synthesis of new transcriptase molecules, because it does not occur in cells treated with protein synthesis inhibitors.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
has no effect on the reaction (Chow & Simpson, 1971). Inhibitors of DNA-dependent polymerases, such as actinomycin D, do not affect the reaction. Maximum activity for the reaction depends on the presence of both Mn w and Mg*+ ions (Carroll, McGeoch & Many, 1975). The levels of enzyme activity associated with different strains of influenza virus were found to vary over an approximately 20-fold range (Chow & Simpson, 1971). However, these differences disappear when reaction mixtures are incubated with 0-3 mM G P G (see below). This suggests that the observed strain differences are due to differences in the initiation of RNA synthesis in vitro, rather than quantitative difference in levels of enzyme between different strains (Carroll et al., 1975). Under optimal conditions, at least 80 % of the genome is transcribed in vitro (Bishop, Roy, Bean & Simpson, 1972; Carroll et al, 1975) but the process is slow and nonrepetitive. The product of the reaction appears to be entirely complementary to vRNA, suggesting that the primary function of this virus-associated enzyme is to synthesize mRNA; replicase activity has not been ascribed to this enzyme. Hefti, Roy & Bishop (1975) found that, although there are multiple initiation sites for the influenza virus transcriptase, the initial sequence for each site is unique, beginning pppGpCp. The activity of the virion-associated transcriptase is stimulated by guanosine and, depending on the strain of virus used, the stimulation of the initial reaction is up to 10-fold (McGeoch & Kitron, 1975). 5'-GMP, 3',5'-cyclic GMP and 5'-GDP show lesser stimulation effects. No other nucleosides or 5'-NMPs have any, but the dinucleoside monophosphates GpG and GpC show large stimulations. Stimulation represents preferential initiation of cRNA synthesis and guanosine is incorporated specifically at the 5' terminus of the progeny RNA. This is the first demonstration of initiation of synthesis of a nucleic acid chain with a nucleoside and suggests that initiation of RNA synthesis by transcriptase might be a suitable target for chemotherapy. The activation effects of GpG and GpC suggest that they too act as chain initiators. It is not known for certain what structural polypeptides constitute the transcriptase, as the enzyme is bound tightly to the nucleocapsid (RNP) structures, but three minor components of the virus designated as polypeptides P^ P 2 and P8, ranging in molecular weight from 85 000-96 000 are always associated with the transcription complex and seen likely candidates as structural components of the enzyme.
Influenza virus RNA
13
Influenza virus messenger RNA
Replication The role of the cell nucleus Any discussion of the replication of influenza virus RNA would be incomplete without a consideration of the role played by cell nuclei in this process. Influenza viruses require the presence of a cell nucleus before any virus-directed RNA or protein synthesis can take place (Follett et al., 1974; Kelly, Avery & Dimmock, 1974). Since these viruses do not multiply in the presence of inhibitors of DNA function such as actinomycin D or mitomycin C (Barry et al, 1962; Barry, 1964; Rott, Saber & Scholtissek, 1965; Nayak & Rasmussen, 1966) or in the presence of a-amanitin (Rott & Scholtissek, 1970; Mahy et al., 1972) it is likely that cellular DNA-dependent RNA synthesis is required for the initiation of successful infection. The possibility that an individual component of the segmented RNA genome of the virus is a cell gene product has been excluded by detailed hybridization studies (Cox & Barry, 1975). The functional significance of cell gene transcription in influenza replication is not yet understood.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
RNA complementary to vRNA has been detected in polyribosome fractions of infected cells and has properties expected of mRNA (Pons, 1972; Etkind & Krug, 1974). In a detailed characterization of influenza mRNA associated with polyribosomes Glass et al. (1975) found that virus mRNA was first detected at 45 min after infection. As the synthesis of total cRNA is just beginning at this time (Bean & Simpson, 1973), nascent cRNA must be rapidly processed into polyribosomal RNA. Virus mRNA was found both on membrane bound and free polyribosomes, it contained poly(A), and reached its maximum rate of appearance at 2-2-5 h after infection. From 45 min onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80 % of the genome RNA, while poly(A)-minus RNA contained sequences complementary to 90-100% of the genome. It has been reported (Skehel, 1973) that some virus proteins are synthesized in detectable amounts earlier in infection than others, implying that part of the genome RNA may be transcribed at an earlier stage of infection than the rest. Data to support this idea have been provided by Avery & Dimmock (1975). In contrast, Glass et al. (1975) found that even at the earliest stages of infection mRNA sequences complementary to more than 90% of the genome RNA were being synthesized. They found, however, that early in infection there was a gross asymmetry in sequence representation, in that almost half the genome appeared to be represented in much greater quantity than the other half. From this they concluded that influenza viruses do not exhibit temporal control of transcription. It has been demonstrated recently that many eukaryotic and viral mRNAs undergo modification of their 5'-termini by methylation, an apparent prerequisite for translation (Shatkin, Banerjee, Both, Furuichi & Nuthukrishnan, 1975). The modified structure at the 5'-terminus is an inverted nucleotide, 7-methylguanylic acid (m'G) and its addition to the mRNA is effected by pyrophosphate linkage and methylation at the 2'-position of the first nucleotide at the 5'-terminus of mRNA in the presence of the methyl-donor S-adenosylmethionine (SAM) (Miura et al., 1975). This process is known as "blocking" or "capping" and has been demonstrated in vitro when the influenza transcriptase product is synthesized in the presence of SAM (Miura et al., 1975). The implication is that influenza mRNA is terminally modified in vivo.
14
R. D. Barry
Virus-specific RNA replication RNA with the characteristics of a base-paired replicative form (Duesberg & Robinson, 1967) and replicative intermediate (Nayak, 1970) have been identified in infected cells. As mentioned above, the formation of cRNA preceeds that of vRNA; synthesis of vRNA reachs a peak at 3 h after infection (Scholtissek & Rott, 1970) and is the predominant RNA synthesized from 4 h onwards (Krug, 1972). The replicase enzyme responsible for the synthesis of vRNA from the cRNA template has not yet been identified, despite an intensive search. Ho & Walters (1966) detected an RNA-dependent RNA polymerase in the microsomal fraction of infected cells and for some time it was assumed that this enzyme was responsible for vRNA synthesis. More recently, an RNA-dependent RNA polymerase was found in highly purified nuclei from influenza-infected cells and reached maximum activity at 3-4 h (Hastie & Mahy, 1973). However, not only are the characteristics of both nuclear and cytoplasmic enzymes very similar, but the product synthesized by both is predominantly cRNA, i.e. both enzymes seem to have the properties of a transcriptase rather than a replicase. At present the nature of the replicase remains obscure, but at least 3 possibilities are worth consideration: (1) that replication and transcription are carried out by the same enzyme; (2) that a presently unrecognized, virus-induced protein acts as the replicase; or (3) that the transcription enzyme is modified by the addition of an extra protein, to form the replicase. If replication and transcription are different aspects of the activity of a single structural entity, it is surprising that the in vitro product of the variously isolated RNA polymerases is invariably cRNA. It may be that conditions in vitro are far from ideal, in that replication is never initiated, or the cRNA may require modification, such as the acquisition of a protein coat, before it can act as a replication intermediate. The alternative possibility, namely that another protein, either singly or in combination with the existing transcriptase, acts as the replicase, seems more likely. Of the virusinduced proteins found in cells infected by influenza virus the most likely candidate would be the non-structural (NS) polypeptide, to which a function has not yet been ascribed. In conclusion, several aspects of RNA synthesis in cells infected by influenza viruses are not yet understood. Cell nuclear activities obviously play an important role in the initiation of RNA synthesis. It is not known why cRNA synthesis predominates early in
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Apart from the need for cell gene transcription, it is likely that events associated with virus-induced RNA synthesis take place in the cell nucleus. It is known for instance that at least 2 virus-induced proteins, the RNP and a non-structural protein, accumulate in infected cell nuclei (Breitenfeld & Schafer, 1957; Dimmock & Watson, 1969). An early report (Scholtissek et al., 1962) suggested that virus particle-associated RNA was made in the nucleus. However, cell fractionation studies revealed the presence of newly synthesized virus RNA in both the cytoplasm and nucleoplasm, although cRNA was predominately cytoplasmic (Krug, 1972). The topographical studies based on autoradiography (Armstrong & Barry, 1974) suggest that major phases of virus-induced RNA synthesis occur in cell nuclei. Nuclei may be important in influenza virus replication for at least 3 reasons. In the first place, RNA synthesis directed by host DNA is needed in the early stages of replication. Following this, nuclei may provide not only the site of viral RNA synthesis, but also the site of assembly of the RNP.
Influenza virus RNA
15
infection, or what factors are responsible for the switch from cRNA to vRNA synthesis. Furthermore, the structural components of either the transcriptase orreplicasehave not been identified directly and it is not known whether either of them require host factors for in vivo activity. The overall interrelationships of viral replication to RNA transcription are a virgin field of study, with important questions yet to be asked concerning host specificity and viral to host interrelationships (Bishop & Flamand, 1975). References
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Andrewes, C. H. & Pereira, H. G. Viruses of Vertebrates. Bailliere Tindall, London (1972), 3rd edition. Armstrong, S. J. & Barry, R. D. The topography of RNA synthesis in cells infected with fowl plague virus. Journal of General Virology 24: 535-47 (1974). Avery, R. J. & Dimmock, N. J. Temporal control of transcription of influenza virus RNA. Virology 64: 409-14 (1975). Baltimore, D. & Franklin, R. M. Preliminary data on a virus specific enzyme system responsible for the synthesis of viral RNA. Biochemical and Biophysical Research Communications 9: 388-92 (1961). Baltimore, D., Eggers, H. J., Franklin, R. M. & Tamm, I. Poliovirus induced RNA polymerase and the effects of virus specific inhibitors on its production. Proceedings of the National Academy of Sciences of the United States of America 49: 843-9 (1963). Barry, R. D. The effects of actinomycin D and ultraviolet irradiation on the formation of fowl plague virus. Virology 24: 398-405 (1964). Barry, R. D., Ives, D. R. & Cruickshank, J. G. Participation of deoxyribonucleic acid in the multiplication of influenza virus. Nature 194: 1139-40 (1962). Bean, W. J. & Simpson, R. W. Primary transcription of the influenza virus genome in permissive cells. Virology 56: 646-51 (1973). Bishop, D. H. L. & Flamand, A. Transcription processes of animal RNA viruses. Control Processes in Virus Multiplication, Society for General Microbiology, 25th Symposium, Cambridge University Press (1975), pp. 95-152. Bishop, J. M. & Levintow, L. Replicative forms of viral RNA. Progress in Medical Virology 13: 1-82(1971). Bishop, D. H. L., Obijeski, J. F. & Simpson, R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. 1. Optimal conditions for in vitro activity of the ribonucleic acid dependant ribonucleic acid polymerase. Journal of Virology %: 66-73 (1971). Bishop, D. H. L., Roy, P., Bean, W. J. & Simpson, R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. Ill Completeness of the transcription process. Journal of Virology 10: 689-97 (1972). Breitenfeld, P. M. & Schafer, W. The formation of fowl plague virus antigens in infected cells as studied with fluorescent antibodies. Virology 4: 328-45 (1957). Carroll, A. R., McGeoch, D. & Mahy, B. W. J. In vitro transcription by the influenza virion RNA transcriptase. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47: 95-102 (1975). Cheyne, I. M. & White, D. O. Growth of paramyxoviruses in anucleate cells. Australian Journal of Experimental Biology and Medical Science 47: 145-7 (1969). Chow, N-L. & Simpson, R. W. RNA dependent RNA polymerase activity associated with virions and sub-viral particles of myxovirus. Proceedings of the National Academy of Science of the United States of America 68: 752-6 (1971). Cooper, S. & Zinder, N. D. The growth of RNA bacteriophage: role of DNA synthesis. Virology 18: 405-11 (1962). Cox, N. J. & Barry, R. D. Hybridization studies of the relationship between influenza virus RNA and cellular DNA. Virology (1975, in press). Davies, P. & Barry, R. D. Nucleic acid of influenza virus. Nature 211: 384-7 (1966). Dimmock, N. J. & Watson, D. H. Proteins specified by influenza virus in infected cells: analysis by polyacrylamide gel electrophoresis of antigens not present in the virus particle. Journal of General Virology 5: 499-509 (1969).
16
R. D. Barry
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
Duesberg, P. H. & Robinson, W. S. On the structure and replication of influenza virus. Journal of Molecular Biology 25: 383-405 (1967). Etkind, P. R. & Krug, R. M. Influenza viral messenger RNA. Virology 62: 38-45 (1974). Flamand, A. & Bishop, D. H. L. Primary in vivo transcription of vesicular stomatitis virus and temperature-sensitive mutants of five vesicular stomatitis virus complementation groups. Journal of Virology 12: 1238-52 (1973). Follett, E. A. C , Pringle, C. R., Wunner, W. H. & Skehel, J. J. Virus replication in enucleate cells: vesicular stomatitis virus and influenza virus. Journal of Virology 13: 394-9 (1974). Glass, S. E., McGeoch, D. & Barry, R. D. Characteristics of the messenger RNA of influenza virus. Journal of Virology (1975, in press). Hastie, N. D. & Mahy, B. W. J. RNA polymerase in nuclei of cells infected with influenza virus. Journal of Virology 12: 951-61 (1973). Hefti, E., Roy, P. & Bishop, D. H. L. The initiation of transcription by influenza virion transcription. Negative Strand Viruses. Academic Press, London (1975), pp. 307-26. Ho, P. P. K. & Walters, P. Influenza virus induced ribonucleic acid nucleotidyl transferase and the effect of actinomycin D on its formation. Biochemistry 5: 231-5 (1966). Kelly, D. C , Avery, R. J. & Dimmock, N. J. Failure of an influenza virus to initiate infection in enucleate BHK cells. Journal of Virology 13: 1155-61 (1974). Krug, R. M. Cytoplasmic and nucleoplasmk viral RNPs in influenza-virus-infected MDCK cells. Virology 50: 103-13(1972). McGeoch, D. & Kitron, N. Influenza virion RNA dependent RNA polymerase: stimulation by guanosine and related components. Journal of Virology 15: 686-95 (1975). Mahy, B. W. J. & Barry, R. D. Negative Strand Viruses. Academic Press, London (1975). Mahy, B. W. J., Hastie, N. D. & Armstrong, S. J. Inhibition of influenza virus replication by a-amanitin: mode of action. Proceedings of the National Academy of Sciences of the United States of America 69: 1421-4 (1972). Montagnier, L. &Saunders, F. K. Replication forms of encephelomyocarditis virus ribonucleic acid. Nature 199: 664-7 (1963). Miura, K., Furuichi, Y., Shimotohno, K., Urushibara, T., Watanabe, K. & Suguira M. Modification at the 5'-terminus of messenger RNA strand. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47: 153-60 (1975). Nayak, D. P. Replication of influenza virus RNA. Biology ofLarge RNA viruses. Academic Press, London (1970), pp. 371-91. Nayak, D. P. & Rasmussen, A. F. J. Influence of mitomycin C on the replication of influenza viruses. Virology 30: 673-83 (1966). Oxford, J. S. Polypeptide composition of influenza B viruses and enzymes associated with the purified virus particles. Journal of Virology 12: 827-35 (1973). Penhoet, E., Miller, H., Doyle, M. & Blatti, S. RNA dependent RNA polymerase activity in influenza viruses. Proceedings of the National Academy of Sciences of the United States of America 68: 1369-71 (1971). Pons, M. Studies on the replication of influenza virus RNA. Virology 47: 823-32 (1972). Reich, E., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. Effect of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134: 556-7 (1961). Rott, R., Saber, S. & Scholtissek, C. Effect on myxoviruses of mitomycin C, actinomycin D and pretreatment of the host cell with ultraviolet light. Nature 205: 1187-90 (1965). Rott, R. & Schottissek, C. Specific inhibition of influenza replication by a-amanitin. Nature 228: 56 (1970). Schottissek, C. & Rott, R. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40: 989-96 (1970). Schottissek, C , Rott, R., Hausen, P., Hausen, H. & Schafer, W. Comparative studies of RNA and protein synthesis with a myxovirus and a small polyhedral virus' Cold Spring Harbor Symposium of Quantitative Biology 27: 245-57 (1962). Shatkin, A. J., Baneriee, A. K., Both, G. W., Furuichi, Y. & Nuthukrishnan, S. 5'-terminal 7-methylguanosine in eukaryotic mRNAs and its requirement for translation. Colloques de Vlnstitut National de la Sante et de la Recherche Medicate (INSERM) 47:177-36 (1975). Skehel, J. J. RNA-dependent polymerase activity of the influenza virus. Virology 45: 793-6 (1971).
Influenza virus RNA
17
Skehel, J. J. Polypeptide synthesis in influenza vims infected cells. Virology 49: 23-6 (1972). Skehel, J. J. Early polypeptide synthesis in influenza virus infected cells. Virology 56: 394-9 (1973). Simon, E. H. Evidence for the non-participation of DNA in viral RNA synthesis. Virology 13: 105-18 (1961). Temin, H. M. Formation and activation of the provirus of RNA sarcoma viruses. Biology of Large RNA viruses. Academic Press, London (1970), pp. 233-49.
Downloaded from http://jac.oxfordjournals.org/ at University of York on July 17, 2015
