BACTERIOLOGICAL REviEWS, Sept. 1971, p, 235-241 Vol. 3S, No. 3 Copyrlsht © 1971 American Society for Microbio10BY Printed In U.S.A. Expression of Animal Virus Genomes
DAVID BALTIMORE Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
INTRODUCfiON ... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 235
CENTRAL ROLE OF MESSENGER RIBONUCLEIC ACID 0 0 0 0 0 0 0 0 0 0 0 0 0 0 235
THE VIRAL GENETIC SYSTEM 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 235
VIRION NUCLEIC ACID POLYMERASES 0 0 0 00 ...... 0 .. 0 ...... 0 .. 0 .. 0 237
POUOVIRUS 0 0 0 0 0 0 ° 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 237
~~··························································~0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 VESICULAR STOMATITIS VIRUS 237
RNA TUMOR VIRUSES 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 238
IMPUCATIONS FOR CELL BIOLOGY 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 238
Protein Synthesis 0 0 ° 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 238
RNA-Dependent Transcription in Cells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 239
RNA-Dependent DNA Synthesis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 239
CONCLUSION 0 0 00 .. 0 .. 00 00 0 .... 0 .... 0 .. 0 .... 0 .. 0 0 .. 0 .... 0 ...... 0 0 0 0 239
LITERATURE CITED 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 240
INTRODUCTION replication and transcription can be considered The world of animal viruses appears to offer the viral genetic system (6). There are two end an unfathomable diversity of specimens but, as products of the viral genetic system: new genetic the molecular biology of the replication of many material and messenger ribonucleic acid viruses has been studied, a pattern of behavior (mRNA). has emerged. The viruses can be divided into The one process which all viruses must perform classes, each of which has its own method of is mRNA synthesis. The central role of mRNA transmitting its genetic information from one derives from the fact that viruses use cellular generation to the next and its own style of ex ribosomes and soluble factors to translate their pressing its genetic information. Although in mRNA. Therefore, the job of the virus is almost some cases the data are still fragmentary it is finished once it has made mRNA, except for possible to outline the behavior of these s;stems certain minor alterations in the functioning of the and to place them in a formal scheme. In this translation machinery. paper I will present such a scheme and I will discuss in some detail the behavior of three viral THE VIRAL GENETIC SYSTEM systems which have been investigated in my The specific mechanism for mRNA synthesis laboratory. Furthermore, I will discuss some of used by a given virus depends on the structure of the implications of the existence of these viral the viral genetic material. Figure 1 is a composite systems in the context of the behavior of normal diagram of the pathways of mRNA synthesis cells. which are used by various classes of viruses. To CENTRAL ROLE OF MESSENGER maintain unity in the diagram, mRNA has been RIBONUCLEIC ACID defined as "+" ribonucleic acid (RNA) for all the systems. This does violation to tradition in Viruses have evolved to the point where they certain of the viral systems, but the gain in order are. concerned mainly with two processes: dupli cation of their genetic material and controlled seems worth the change of previous definitions. expression of the information inherent in the The data on which this diagram are based genetic material. The specific mechanisms which are fairly conclusive for all cases except class VI. a given virus uses to carry out the processes of A general discussion of each class follows. 235 236 BALTIMORE BACfERIOL. REV.
+DNA reason for including them in this category. In order for the class IV viruses to synthesize mRNA ~ ~® (or more virion RNA), they must first produce a +RNA--DNA- ±DNA template " " strand of RNA (2). ~ ~CD ® Class V +RNA-- RNA -I+ m RNA I -±RNA Class V consists of viruses which have a single stranded RNA genome which is complementary -RNA in base sequence to the messenger RNA. Synthesis of their mRNA therefore involves transfer of in FIG. 1. Examples: I = T4 phage, vaccinia virus; II = if>X/74; Ill= reovirus; IV= RNA phages, polio formation from one single-stranded RNA to its virus; V = vesicular stomatitis virus, Newcastle disease complementary RNA (17, 25). Vesicular stoma virus; VI = RNA tumor viruses. titis virus (VSV) is representative of this group (25), but Newcastle disease virus (NDV; refer Class I ence 17) and possibly influenza (18) are also members. This group may then include all of Class I consists of all viruses which have a the rhabdo-, paramyxo-, and myxoviruses. If double-stranded deoxyribonucleic acid (DNA) the myxoviruses are, in fact, part of this class, genome. These viruses, like cells, carry out an then some members have a single RNA molecule asymmetric transcription of their DNA to give as their genome which is transcribed into partial rise to mRNA. Because in many viral systems replicas (VSV and NDV; references 17, 25), different mRNA species come from different whereas others have multiple pieces of genome DNA strands, the designation of " +" and " - " RNA (influenza; reference 20). strands in the genome is not meaningful, although locally the designation is possible. Class VI
Class II Class VI consists of the RNA tumor viruses and other viruses which have a DNA interme Class II consists of viruses which have a single diate in their growth and a single-stranded stranded DNA genome of the same polarity RNA genome. To fit these viruses into the as the mRNA (23, 38, 40). To my knowledge, scheme, it has been necessary to stretch the avail no virus with single-stranded DNA comple able evidence a long way, because the intra mentary to mRNA has yet been found, although cellular DNA is still more of a postulate than a viruses with strands of both polarity in separate reality and the mRNA has not yet been charac particles have been characterized (19, 33, 37). terized. However, the evidence from metabolic inhibition studies (1, 44, 45) and the existence Class III of the RNA-dependent DNA polymerase in the Class III consists of viruses which have a virions of these viruses (3, 46) strongly implies a double-stranded RNA genome. The mRNA for DNA intermediate. Furthermore, the easiest these viruses represents an asymmetric transcript (but not necessarily the correct) scheme for the of the genome (31). The known viruses of this handling of this DNA would be an asymmetric type have multiple pieces of double-stranded transcription to give rise to both virion RNA RNA, each of which apparently contains the and mRNA. For this reason, I suggest that the information for synthesis of a single protein two types of RNA probably have the same rather (41). than complementary base sequences. There are many viruses about which so little Class IV is known that they cannot be placed in the scheme Class IV consists of single-stranded RNA in Fig. 1. However, as their transcription becomes viruses Vlbme mRNA is identical in base se understood, either they should fall into place quence to virion RNA. In the case of poliovirus, in a recognized pattern or new classes will have the mRNA appears to be identical to the virion to be added. RNA in both size and base sequence (2), and The overall viral genetic systems as defined it would appear that all of the picornaviruses above consist of both the transcription and repli share this property. The arboviruses (or toga cation systems (6). Figure 1 omits replication viruses) probably fall into this class also, al because, except for the systems where replica though their mRNA has not been precisely tion and transcription are synonomous (class defined. As noted below, the ability to isolate IV and maybe VI), little is known about repli infectious arbovirus nucleic acid ( 48) is one cation of the virion RNA. Viruses with similar VoL. 35, 1971 EXPRESSION OF ANIMAL VIRUS GENOMES 237 transcriptional systems could have different mented, and the nature of some of the interme replicational systems leading to the necessity diate molecules has bee!l investigated (see to extend the class designations. reference 2), but the lack oi an in vitro system in which the molecular details can be analyzed has VIRION NUCLEIC ACID POLYMERASES frustrated progress. One important aspect of the transcriptional One aspect of the poliovirus genetic system systems of animal viruses has emerged during which has led to some surprising results is the the last few years: the role of virion-associated existence of a single mRNA molecule which is nucleic acid polymerases. The initial discovery the template for synthesis of a number of viral of the virion-associated DNA-dependent RNA polypeptides (2, 5). The initial discovery of this polymerase of vaccinia virus (30, 35), followed situation was unremarkable because it was as a year later by the discovery of the double sumed that the mRNA represented a polycis stranded RNA-dependent RNA polymerase of tronic mRNA in the sense in which such mole reovirus (16, 39), opened up a new concept of cules are understood in bacteria. However, anal how viruses carry out their initial transcrip ysis of protein synthesis in infected cells has shown tional processes. More recently, the finding that that a heretofore unknown mechanism is opera VSV (8) and NOV (24) both have virion-asso tive. The mRNA encodes a single polypeptide ciated RNA-dependent transcriptases has led chain of about 250,000 daltons which is cleaved us to see such enzymes as the rule rather than by proteolytic enzymes to form the functional the exception. Finally, the existence of an RNA viral proteins (5, 7, 26, 28). The evidence for this dependent DNA polymerase in the virions of the model has been presented in detail elsewhere RNA tumor viruses (3, 46) has led us to the con (5, 7, 26, 28) and need not be reiterated here, ex cept that, whenever the first function performed cept to note that two different types of experi by virion nucleic acid after its introduction into ments have revealed the single, large precursor the cell is the transfer of its information to another protein: exposure of infected cells to amino acid nucleic acid, the enzyme responsible for this analogues and incubation at high temperature. transfer is likely to be found in the virion. The cleavage of this large polypeptide pro The discovery of virion polymerases has pro ceeds in at least three steps. First, smaller poly vided a rationale for an old puzzle in virology. peptides are cleaved from the single, large poly Why is it that infectious nucleic acid can be ex peptide while it is in the process of synthesis; tracted from the virions of some viruses but not these can be called nascent cleavages (26). Their others? Virion polymerases provide the clue to existence explains why the large polypeptide is this puzzle; where they exist the nucleic acid is never seen in untreated, infected cells: it never noninfectious, where the nucleic- acid is infec really exists in such cells because it is cut up be tious they could not exist or at least they could fore it is finished. The second class of cleavages not serve an obligate role. The infectivity of arbo involves processing· of the initial products and virus RNA was the major reason for including occurs more slowly. Scission of the single capsid these viruses in class V; the infectivity of their precursor molecule into three pieces typifies this RNA suggests that virion RNA and mRNA series of events (26, 28). The capsid precursor must be of the same strand of RNA for these has a half-life of about 15 minutes. A third type viruses. of cleavage is known for which only a single ex ampie has been seen. One of the capsid proteins POLIOVIRUS is cleaved into two pieces in an event which occurs The foregoing generalities provide a frame as RNA is being combined with viral protein to work for considering the molecular biology of form the virion (27). The immediate precursor of different types of viruses. In the case of polio the virion is, we believe, an icosahedral structure virus, which is the prototype of the group of (the procapsid) consisting of 60 subunits, each of viruses which is understood in m:>st detail the which is made of three proteins (5). As RNA is ptcornaVll'uses,. . the fundamental problems which' added to this structure to make the virion, one of are posed by the scheme in Fig. 1 are the mecha the polypeptides in each of the 60 subunits is nism of synthesis of the complementary RNA cleaved. This cleavage occurs only when RNA is (the "-" stran:i) and the mechanism of syn added to the procapsid; in the abser.ce of RNA thesis of virion RNA. These two problems de synthesis n:> cleavage is observed (27). fine the mechanism of the picornavirus genetic system because they encompass both transcrip VESICULAR STOMATITIS VIRUS tion and replication. The existence of " +" and Recognition of the existence of the class V " " strand synthesis in cells has been docu- viruses came from two types of experiments. The 238 BALTIMORE BACIERIOL. REV. first was the purification from VSV -specific poly plates (34, 36, 42, 43). To investigate the questions ribosomes of mRNA molecules which were much which we posed, it was necessary to use homo smaller than the 4.2 X lOS-dalton single-stranded polymer templates rather than true nucleic acids, virion RNA and which would quantitatively hy because their behavior was more easily controlled. bridize to virion RNA (25). The second was the Because most of the data have been recently demonstration of the virion RNA polymerase published (9, 11), I present the following conclu which would carry out the synthesis of small RNA sions of this study. molecules from a long single-stranded template (i) The enzyme cannot start molecules de novo (8). Together these experiments indicated that the but requires at least an oligonucleotide primer to VSV transcription system was different from initiate synthesis. In this property, the enzyme is those which were previously known. The exist like other known DNA polymerases (14, 15, 21, ence of large amounts of intracellular NDV RNA 22) and unlike RNA polymerases, which are able complementary to and smaller than NDV virion to start synthesis with merely a nucleoside tri RNA (17) suggested that it was closely related to phosphate initiator (32). VSV, and the recent discovery of an NDV virion (ii) The enzyme prefers ribohomopolymers to transcriptase (24) has fortified that conclusion. deoxyribohomopolymers, but its preference is not The very recent identification of an influenza absolute (see Table 1 and reference 9). It will virion RNA polymerase (18) indicates that the utilize poly(dC) as a template if an oligo(dG) is myxoviruses may belong to the same class, al provided to initiate synthesis, but no other deoxy though little is known about their mRNA. ribohomopolymer will act as template even if the appropriate primer is provided. In contrast, Esch RNA TUMOR VIRUSES erichia coli DNA polymerase I (the "Kornberg enzyme") shows a preference for deoxyribohomo As mentioned above, the genetic system of the polymers over ribohomopolymers although, RNA tumor viruses is not known, but a reason again, one ambiguity is seen: the enzyme will able guess as to its nature is possible. There are utilize both poly(rA) and poly(dA). two classes of evidence which point to a DNA intermediate. One is the data accumulated by IMPLICATIONS FOR CELL BIOLOGY using metabolic inhibitors and other perturba tions of events in cells (1, 44, 45). The measured The foregoing material shows that we are in a variable in these experiments is production of position now to understand viral genetic systems. virions or morphological transformation, or both, It is necessary to stretch evidence in places and criteria which are distant from the affected to make some guesses in order to provide unify biochemical process and which have therefore ing concepts at the present time, but the rate of provided circumstantial but not convincing evi progress is so great right now that within a few dence. The second class of evidence is the dem years the picture should be quite clear. It is im onstration of an RNA-dependent DNA polym portant that investigation of such systems con erase in virions of the RNA tumor viruses (3, 46). tinue because the results have implications be This has been taken as strong evidence for a DNA yond the province of virology. I should like to intermediate, but, in fact, it shows only how the look at some of these implications if for no other DNA intermediate could be formed if it is formed. reason than to illustrate how virology can illumi A third form of evidence, and potentially the most nate cell biology. I will mention three examples powerful, would be the direct demonstration of drawn from the three areas of virology described the postulated DNA in infected cells; however, above. aside from a few suggestive reports (12, 13), strong evidence has yet to appear. Protein Synthesis Recently, my laboratory has been focusing on When we first realized that poliovirus proteins two aspects of the behavior of the virion DNA were all derived from a single precursor, we asked polymerase in RNA tumor viruses. (i) Does the the obvious question: Why does the virus use this enzyme start DNA molecules de novo or does it apparently clumsy mechanism? We suggested, as require a primer? (ii) Is the enzyme really an a possible answer, that perhaps all mammalian RNA-dependent DNA polymerase, that is, does mRNA is like poliovirus RNA, i.e., it has only a it prefer to use ribonucleic acid templates rather single initiation site for protein synthesis and only than deoxyribonucleic acid templates? These a single termination site (28). Many results in the questions could be investigated because the RNA literature were consistent with this idea (see refer tumor virus DNA polymerase, as opposed to all ence 28 for further discussion), and as yet no con other known virion-associated nucleic acid polym tradictory evidence has appeared. A corollary of erases, will utilize added nucleic acids as tern- this idea is that operons could not exist in mam- VoL. 35, 1971 EXPRESSION OF ANIMAL VIRUS GENOMES 239
TABLE 1. Template specificity of DNA polymerasesa
Homopolymers• Enzymeb rA rC ri rU dA dC dl dT ------AMV DNA polym- +++ +++ ++ 0 0 ++ 0 0 erase [ MLV DNA polym- +++ ++ + ++ 0 +++ 0 0 erase [>poly(rC)] Escherichia coli + 0 0 0 +++ ++ + ++ DNA polymerase I [ a Relative activities of different templates for the viral enzymes were estimated from the data in Fig. 3 and 4 in reference 9 and from other experiments in which rates were determined by using high specific activity precursors. Where active polymers are designated+++ or++, inactive polymers (0) had less than 1% of the activity of their active homologues. The data on E. coli DNA polymerase I were obtained with a highly purified preparation (fraction 7; reference 30) kindly provided by Arthur Kornberg and assayed with 0.05 M potassium phosphate buffer (pH 7.4) and 6 mM magnesium acetate (10). Templates, primers, and substrates were varied as in the assays of the viral DNA polymerases. Poly(dA) was 10-fold more active than poly(rA) as a template for the E. coli enzyme. b Abbreviations: AMV, avian myeloblastosis virus; MLV, murein leukemia virus. • Abbreviations: Homopolymers of ribuadenylate (rA), ribucytidylate (rC), riboinosinate (rl), ribouridylate (rU), deoxyadenylate (dA), deoxycytidylate (dC), deoxyinosinate (dl), and deoxythymidylate (dT). malian cells as they do in bacteria, as units of enzyme and such a process in uninfected cells. It transcription containing the information for is possible to imagine such systems having a role linked but separate units of translation. in the processes of differentiation, the workings of the immunological system, and in the mech RNA-Dependent Transcription in Cells anisms of memory storage (4). Evidence that The mechanism of mRNA synthesis used by ribosomal gene amplification involves RNA-de VSV and the other class V viruses suggests an pendent DNA synthesis has already appeared intriguing possibility for formation of some (47). mRNA in cells. Possibly the DNA genome is transcribed into anti-mRNA which, in turn, is CONCLUSION transcribed into mRNA. Although the postula Viruses which have a double-stranded DNA tion of an intermediate RNA may seem gratui genome can express and duplicate their genetic tous, it would (i) provide a role for the unstable material by processes which are at least formally nuclear heterogeneous RNA; (ii) provide a new identical to those used by cells. Viruses with other level for control of gene expression and a new types of genomes require special systems for repli mechanism for quantitative regulation of mRNA cation and transcription. These processes make synthesis; and (iii) provide an explanation for the up the viral genetic system, and it is possible to origin of some if not all RNA viruses. Evidence group viruses according to general properties of for such a mode of information transfer in cells their genetic systems. A central element in the could come from hybridization of mRNA to nu genetic systems of many viruses is a polymerase clear RNA. Experiments in progress indicate that carried in the virion which carries out a transfer in HeLa cells there is at least limited complemen of information from one type of nucleic acid to tarity between these two species of RNA (M. another as probably the earliest event in viral Stampfer, M. Rosbash, A. S. Huang, and D. replication. Baltimore, unpublished data). A comparison of gene expression in three viral systems indicates the diversity of behavior which RNA-Dependent DNA Synthesis exists. The picornaviruses encode multiple pro Discovery of the RNA-dependent DNA polym tein species in a single polypeptide. The rhabdo erase in RNA tumor viruses has touched off an viruses and paramyxoviruses carry many genes in explosion of interest in the possibility of such an a single RNA but synthesize separate, comple- 240 BALTIMORE BACI'ERIOL. REV. mentary RNA species. The RNA tumor viruses synthesis in cells infected with Newcastle disease virus. J. probably transfer their genetic information from Mol. Bioi. 23:1-21. 18. Chow, N-L., and R. W. Simpson. 1971. RNA-dependent RNA RNA to DNA, where it can be expressed by polymerase activity associated with virions and subviral DNA-dependent RNA synthesis. particles of myxoviruses. Proc. Nat. Acad. Sci. U.S.A. 68: Each of these examples suggests the existence 752-756. of processes in cells which otherwise might not 19. Crawford, L. V., E. A. C. Fallett, M. G. Burdon, and D. J. McGeach. 1969. The DNA of a minute virus of mice. 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