Viruses with Segmented Ribonucleic Acid Genomes: Multiplication of Influenza Versus Reovirus

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1 BACTERIOLOGICAL REVIEWS, Sept. 1971, p Copyright 1971 American Society for Microbiology Vol. 35, No. 3 Printed in U.S.A. Viruses with Segmented Ribonucleic Acid Genomes: Multiplication of Influenza Versus Reovirus A. J. SHATKIN Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey INTRODUCTION VIRION STRUCTURE GENOME RNA GENETICS REPLICATION CYCLE Adsorption and Penetration RNA Synthesis Reovirus single-stranded RNA Reovirus genome RNA Influenza RNA Protein Synthesis Reovirus 260 Influenza Maturation CONCLUSIONS 262 LITERATURE CITED INTRODUCTION multiplication in a manner analogous to the assortment of chromosomal linkage groups (143). More than 10 years ago it was proposed that the genome of influenza virus is made up of two or In addition to a high frequency of recombination, more single-stranded ribonucleic acid (RNA) influenza shares with reovirus other biological segments rather than one continuous chain of properties which are consistent with the organization of their genomes into functional segments. covalently linked nucleotides (26, 60). The first evidence for such an unusual viral genome came These include multiplicity reactivation (10, 94) from genetic studies. These were later supported and the formation of noninfectious particles deficient in one of the genome segments (27, 105, by the finding that RNA extracted from influenza and other myxoviruses consists of a 110). For this review, the extensively studied mixture of RNA fragments of molecular weight myxoviruses (influenza) and reovirus groups have lower than that of the total viral RNA estimated been selected as representative examples of chemically (3, 13, 38, 40a, 101, 109). It is now viruses with segmented RNA genomes. Their known that the double-stranded RNA genomes composition and structure are compared, and the of reoviruses and similar animal and plant viruses possible effects of genome fragmentation on virus are made up of distinct segments (47, 48, 70, 134, multiplication are considered. 135, 156). Recent studies of a variety of singlestranded RNA viruses including paramyxoviruses VIRION STRUCTURE (Newcastle disease virus, NDV; reference 75), Purified influenza virions are 100-nm spherical rhabdoviruses (vesicular stomatitis virus, VSV; particles when observed in the electron microscope by the techniques of shadowing and negative references 67, 97, 104, 121), arboviruses (Sindbis virus; reference 36), and the RNA tumor viruses staining. More recent examinations by the freezedrying method revealed that they are icosahedral (4, 40) have shown that viral RNA genomes, or their complement in infected cells, are often found in shape (103). In some strains, filamentous forms as subunits. Genetic and biochemical studies also occur. The particles contain about 20% indicate that the RNA fragments are subgenomic lipid, 70% protein, 1% RNA, and 10% carbohydrate by weight (19, 46). Virions consist of a structural units which have functional significance. For example, the recombination frequency predicted for an intact molecule of the size of the capsid, a complex of RNA and protein often lipid-containing envelope surrounding the nucleo- influenza genome is exceeded by several-fold observed in a helical configuration in electron experimentally, suggesting that the influenza micrographs (114). A predominant polypeptide RNA segments may be exchanged during virus among the several found in virions is the internal 250

2 VOL. 35, 1971 VIRUSES WITH SEGMENTED RNA GENOMES 251 protein which comprises 85 to 90% of the ribonucleoprotein complex (RNP; reference 165). It is an arginine-rich polypeptide (65) having a molecular weight of 50,000 to 60,000 daltons as determined by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels (30, 31, 42, 59). Presumably, it provides the structural backbone for packaging the RNA within the virion (111). Complement-fixing, group-specific antigenicity (S antigen) also resides in the RNP (114). The lipoprotein membrane which encloses the RNP contains the type-specific antigen which may be related to a major structural polypeptide having a molecular weight of about 20,000 found in this component of the virion (131). Attached to the membrane on the surface of the particle are two biologically active substructures: the hemagglutinin (HA) and the neuraminidase (NA). Both are high-molecular-weight glycoproteins (-150,000 daltons) composed of subunits. The subunit glycopeptides of the HA and NA have molecular weights of about 50,000 and 60,000 daltons, respectively (163). Host-dependent molecular-weight variations of about 10% have been observed for the glycoproteins but not the carbohydrate-free viral structural polypeptides (30, 58, 131). The surface structures can be removed by proteolytic digestion of virions, resulting in noninfectious, RNP-containing core particles (30, 131). Recently, RNA polymerase activity has been found associated with the cores prepared from purified influenza virus (28). In contrast to the more complex myxoviruses, purified reoviruses have no lipid envelope and are ether-resistant (120). They contain 15% RNA and 85% protein packaged in icosahedral particles about 70 nm in diameter. Reoviruses are considerably more stable than-myxoviruses, possibly as a consequence of their double capsid structure. The outer protein layer can be removed from the inner core particle by digestion of the virion with chymotrypsin and other proteolytic enzymes or by treatment with pyrophosphate or urea (34, 93, 147, 148). The resulting subviral particle (SVP) contains the double-stranded RNA and in some strains may be infectious (148). Three major polypeptides having molecular weights of 155,000, 140,000, and 38,000 daltons estimated by SDS-polyacrylamide gel electrophoresis are present in the SVP (7, 90, 146). The outer capsid layer has four polypeptides having molecular weights of 80,000, 72,000, 42,000, and 34,000 daltons. The seven reovirus structural proteins have been classified into three size groups: X (140,000 and 155,000),, (72,000 and 80,000), and a (34,000, 38,000, and 42,000) (146). Reoviruses contain a group-specific, complement-fixing antigen and a serotype-specific reactivity (115). The whole virion and the SVP both hemagglutinate human erythrocytes (147, 149), but no reovirus structural glycoproteins have been reported. Associated with the SVP are at least two enzyme activities: an RNA polymerase which transcribes all segments of the double-stranded genome (7, 22, 87, 137, 145) and phosphatase(s) which hydrolyzes nucleoside triphosphates to diphosphates (9, 23, 71). Since the procedures presently used to solubilize the virion proteins also result in their denaturation, it is not yet known which polypeptides correspond to the various biological and enzymatic activities. GENOME RNA On the basis of the chemical composition and particle weight of purified virions, the average molecular weight of influenza virus RNA has been estimated to be 2 X 106 to 3 X 106 daltons (1, 19). More recently, values of 4 X 101 to 5 X 101 daltons have been suggested from studies on the molecular weights and number of polypeptides found in purified virus and infected cells (30). In view of the host-related variations in the molecular weights of the glycoproteins and the presence of nonstructural, virus-induced proteins in infected cells (35), it is not possible at present to establish accurately the total daltons of viral proteins and the corresponding molecular weight of RNA required for coding. In fact, it has been suggested recently that the major viral structural proteins arise by post-translational cleavage of a giant precursor polypeptide (42). A direct measure of the genome size is difficult to obtain because purified influenza preparations often contain a high proportion of RNA-deficient, noninfectious particles (von Magnus virus; references 27, 110, 157). Furthermore, RNA isolated from purified influenza virus is not reproducibly infectious, and attempts to demonstrate rescue of biological activity of isolated influenza RNA by adding another strain of influenza virions as a helper have also failed (42). Single-stranded RNA of the average size of the influenza genome would be expected to have a sedimentation constant greater than 30S in solutions containing 0.1 M NaCl. However, in most studies, the RNA extracted from purified virus with phenol and SDS consists of a mixture of smaller molecules (19, 106). The extracted RNA can be resolved into at least five distinct RNA segments in the size range 0.2 X 106 to 0.7 X 106 daltons by electrophoresis in polyacrylamide gels (38, 109). Several distinct double-stranded RNA molecules of the same length as the viral RNA segments have also been obtained from influenza-

3 252 SHATKIN BACrERIOL. REV. infected cells (19, 102, 108). Recently, it was found that by using longer gels for electrophoretic separation many more than five pieces of influenza RNA can be resolved (42). In this study in contrast to reports by other workers, the size distribution of the fragments was found to be dependent upon the strain of virus, type of host cell, and method of infection used. Thus, the possibility must be considered that, under certain experimental conditions, the isolated fragments may arise by breakage during extraction of the viral genome. Further evidence for this possibility has recently come from observations with the electron microscope (89). If one assumes that infectious influenza particles contain the equivalent of one of each of the five RNA segments which are usually resolved in polyacrylamide gels, the genome would have a molecular weight of about 3 X 106 daltons (106). Electron micrographs of RNA carefully extracted from three strains of purified influenza virus by the phenol- SDS method have revealed molecules having a mean length of 2.4 to 2.7 Mim, corresponding to molecular weights of 2.5 x 106 to 2.9 X 106 daltons (89). After brief exposure to ph 3 or storage at 4 C for several days, the long molecules were replaced by RNA pieces of five different sizes but, unexpectedly, with unequal frequency. A fraction of the RNA from one strain had a length corresponding to a molecular weight of 3.6 X 106 daltons, and it was suggested that it represented the entire genome. This work needs to be confirmed since some RNA-protein complexes are known to resist strong denaturing agents including phenol and SDS (80; M. Nomura, personal communication). The implication from the morphological studies (89) that the influenza genome is a single, continuous RNA molecule which breaks during extraction is in sharp contradiction to the occurrence of distinct double-stranded RNA molecules in influenzainfected cells and to biochemical analyses of the 5'-termini of the isolated genome fragments. All RNA segments from purified influenza virus, like those from reovirus (8), seem to contain a unique 5'-terminal nucleotide (164). The presence of 5'-pppAp in the influenza RNA pieces indicates that the fragments do not arise by cleavage of a long RNA molecule but are probably synthesized individually and subsequently packaged into mature influenza virions. The long molecules of viral RNA seen in the electron microscope would then consist of fragments linked end-to-end rather than covalently. Double-stranded RNA isolated from purified reoviruses also consists of a noninfectious mixture of fragments. The fragment sizes are extremely reproducible and differ from those expected for RNA pieces arising from a larger molecule by shearing or random nuclease degradation (18, 139). The reovirus fragment patterns are not altered by varying the virus strain, the host cell type, or the virus purification procedure. Several extraction procedures, including the phenol-sds method which results in infectious RNA from picornaviruses, yield the same pattern of noninfectious RNA segments from purified reovirus and from infected cells. The different RNA fragments do not cross-hybridize and are therefore unique segments without long, common nucleotide sequences (16, 159). The results of hybridization experiments with influenza RNA segments also indicate that they have different base sequences (19). The average RNA content per reovirus particle is greater than 107 daltons (54), but the isolated fragments are considerably smaller. They fall into three size categories on the basis of sedimentation analysis: L (large), 2.4 X 106; M (medium), 1.4 X 106; and S (small), 0.8 x 106 daltons. By electrophoresis in polyacrylamide gels, the mixture is resolved further, and the double-stranded RNA isolated from reovirus type 3 can be separated into three L, three M, and four S fragments (139). In contrast to most preparations of influenza which have a low specific infectivity due to the presence of von Magnus virus, reovirus preparations have been obtained in which essentially all particles are infectious (113, 148). After repeated passage at high multiplicities, purified reovirus preparations include particles lacking the largest of the double-stranded segments (105); the defective particles are noninfectious and can be considered analogous to the von Magnus particles of influenza virus described below (157). These results indicate that all 10 RNA segments are present in reovirus particles and are required for infectivity. The total molecular weight of the double-stranded genome consisting of three L, three M, and four S pieces is about 15 X 106 daltons. There are a number of biological findings which support the hypothesis that the multiple RNA fragments isolated from purified influenza and reovirus are genome subunits of functional as well as structural significance. The infectivity of influenza resides in single virions containing several RNA fragments and is not a cooperative process among several particles each containing a different RNA segment. This is evident from the finding that the loss of plaques observed at increasing dilutions or upon exposure to gamma rays both follow single-hit kinetics (10, 42). In addition, influenza preparations with a plaqueforming unit (PFU) to particle ratio close to one have been reported (88). Chemical inactivation

4 VOL. 35, 1971 VIRUSES WITH SEGMENTED RNA GENOMES 253 of influenza infectivity by treatment with ethylene iminoquinone is accompanied by a sequential loss in the capacity to form hemagglutinin, followed by neuraminidase and finally RNP-antigen (127). Similarly, ultraviolet (UV) treatment of influenza virus inactivates most quickly the capacity to induce synthesis of the highest-molecularweight species of viral RNA and proteins (69). The sequential inactivation of different viral functions is consistent with a subunit arrangement of the genome. Infection of most cell types with influenza virus at a multiplicity greater than 1 PFU per cell leads to the formation of increasing amounts of incomplete virus, the von Magnus effect (157). With each successive undiluted passage, the resulting particles have a reduced capacity to form infectious virus followed by a loss in the ability to elicit HA, NA, and internal antigen production. The order of disappearance in biological activity is the same as that observed with virions inactivated by chemical and physical methods. The von Magnus particles contain a normal spectrum of proteins (110); consequently, they are immunogenic and also interfere with the replication of infectious virus (118). However, they contain a reduced amount of RNA (2). Since they are genetically defective, i.e., do not support multiplicity reactivation, it was suggested that the incomplete particles are deficient in a defined region of the RNA genome (117). It has now been demonstrated that the largest RNA segment is markedly reduced in von Magnus virus as in the defective particles obtained after serial, undiluted passage of reovirus (27, 38, 105, 110). Perhaps the largest RNA segment is the last to be completed in infected cells and is therefore most readily lost under conditions of abortive replication (19, 118). The RNA extracted from influenza-infected cells includes a mixture of virus-specific singlestranded and double-stranded RNA molecules (40a, 102, 108). The latter correspond in length to the single-stranded influenza genome segments, and after denaturation the two types of RNA comigrate in polyacrylamide gels (108). Annealing studies indicate that the single- and doublestranded molecules which are the same length are also homologous in base sequence (19). Presumably, the double-stranded pieces are replicative forms of the single-stranded genome segments, (102). Similarly, RNA extracted from type 3 reovirus-infected cells consists of a mixture of the 10 double-stranded genome fragments and singlestranded RNA species corresponding in length and in base sequence to one strand of each of the duplex molecules (16, 51, 112, 136, 161). The single-stranded reovirus RNA species are found in polyribosomes and probably function as viral messenger RNA species. A comparison of the size of the reovirus structural proteins and the single-stranded RNA species suggests that each functions as a monocistronic messenger. On the basis of a triplet code and an average molecular weight per amino acid of 100, single-stranded RNA species transcribed from the L, M, and S genome segments could code for proteins having approximate molecular weights of 130,000, 70,000, and 40,000, respectively, values close to those determined for the three size groups of reovirus structural proteins. Although these and other genetic and biochemical findings discussed below clearly indicate that the influenza and reovirus RNA fragments represent functionally important genome subunits, it is not known how they are correctly segregated during infection and arranged within infectious particles. If the large number of genome subunits are independently synthesized in influenza and reovirus-infected cells, it is difficult to conceive of a mechanism for their correct assembly into mature virions. It has been pointed out that a high proportion of infectious influenza virions would result from random packaging of RNA fragments, assuming that five fragments are required for infectivity and seven pieces are incorporated into each particle (29, 75). For reoviruses, the higher number of genome segments reduces the probability that random packaging could account for an observed particle to PFU ratio of one. An obvious possibility is that the subunits are weakly linked within the virion and remain together as a replicating complex during the virus multiplication cycle. Recently, it was reported that the reovirus parental genome is conserved and remains associated with structural proteins as a subviral particle throughout infection (141). However, in this study, the proportion of virions which were infectious was not determined. Since particle to PFU ratios of 10 to 100 are commonly obtained, and genetic studies indicate that the noninfectious particles do not participate in the replicative cycle (45), most of the input particles under study were probably not involved in a productive infectious cycle. The genome segments within the virions could be joined by base pairing at their termini or by basic protein "linkers." For influenza virus, it seems more likely that linkage is mediated by the protein portion of the RNP complex acting as a structural backbone for the linear arrangement of the RNA subunits. A continuous filament of RNP is released from some particles when influenza virus is lysed directly on grids for electron microscopy. However, the bulk of the RNP extracted from influenza by treatment with deter-

5 254 SHATKIN gents or ether is a mixture of smaller, distinct components (39, 64, 76). This is in contrast to the RNA of paramyxoviruses (also known as subgroup II myxoviruses) which is extracted from the virion as an intact helix containing the RNA genome as a single molecule having a molecular weight of about 7 X 106 daltons (75). The size of the individual influenza RNP molecules reflects the molecular weight of their constituent RNA segment (39, 76, 106). RNA extracted from the large, 70S RNP components is exclusively the largest segment which has a molecular weight of 0.7 x 101 daltons. Similarly, the RNP components of intermediate size contain the three intermediate-sized RNA species, and the slowly sedimenting RNA complexes have the small genome fragments. All of the RNP molecules contain many molecules of the arginine-rich internal polypeptide. This basic protein apparently provides the structural configuration of the RNP, since after removal of the RNA by ribonuclease digestion or displacement with another polyanion, polyvinylsulfate, the resulting protein retains the shape and sedimentation characteristics of RNP (111). Thus, each RNA segment in the isolated influenza RNP mixture is probably associated with a protein structure of comparable length which consists of many molecules of the argininerich polypeptide. As suggested by Pons (106), the RNP may be one long, folded protein structure with the genome subunits arranged along its length. The intervals between the ends of the RNA segments could represent weak points of folding in the RNP filament which break during isolation. In some instances (89), the deproteinized RNA segments may be held together by small amounts of residual internal polypeptide tightly bound to the ends of the RNA molecules. Since the RNA of RNP is susceptible to ribonuclease but protects the structural protein from protease digestion, the RNA is probably ordered around, rather than within, the internal protein (39, 76, 106). Electron microscopic and chemical studies of reovirus suggests that its genome, like influenza virus, is organized into weakly linked segments within the infectious particle. Double-stranded RNA isolated from virions by a variety of procedures is a mixture of short fragments which have a trimodal length distribution when examined in the electron microscope by the protein monolayer technique (41, 53, 56, 77, 155). The peak lengths of 0.3, 0.6, and 1.1 jm correspond to the L, M, and S genome subunits, respectively. In addition, a few much longer molecules are present. Their number can be increased by using gentler extraction procedures, for example, brief exposure to urea at 4 C or direct spreading of BACrERIOL. REV. virus suspensions on a solution of sodium perchlorate. The longest molecules observed are about 7 gm, the value expected for a duplex genome having a molecular weight of approximately 15 X 106 daltons (155). Millward and Graham compared the number of 3'-terminal nucleoside residues in reovirus double-stranded RNA before and after extraction from the virion (95). They reasoned that if the virion contains 10 distinct segments of RNA, the number of free 3'-OH ends in the viral genome in situ should remain unchanged after extraction of the RNA. Conversely, if the genome is a continuous duplex containing nine specific breakage points, the number of 3'-termini would be greater by as much as 10-fold in the isolated RNA. The purified virions were treated at ph 5.5 with periodate to oxidize the RNA in situ, and the resulting dialdehydes were reduced with 3Hborohydride to label the 3'-ends. A comparison of the radioactive products with RNA which was labeled in an identical manner but after isolation from virions revealed that the double-stranded RNA within the virion contained approximately the same number of 3'-termini as the extracted RNA. Assuming that only 3'-terminal bases and not internal positions were labeled and that the chemical procedures themselves did not result in breakage of the genome, these results indicate that the genome is a discontinuous structure within the virion. Direct analysis of 32P-labeled reovirus double-stranded RNA revealed that all 10 genome segments have a unique 5'-terminal nucleotide, ppgp (8). These findings, together with the microscopic observations, demonstrate that the reovirus genome consists of individually synthesized segments which are linked by weak, noncovalent bonds inside the virion. A basic protein analogous to that of influenza RNP has not been identified in reovirus, but it seems likely that the segments are held together by RNA-protein interactions. It was reported that the 5'-termini of the double-stranded segments have short, single-stranded "tails" of purines which could interact with a protein to provide a basis for joining the RNA segments end-to-end (96). However, direct analysis of the genome segments failed to confirm the presence of such "tails," and the similarity in the 5'-ends of genome RNA and reovirus messenger RNA (mrna) made in vitro strongly suggests that the segments are perfect duplexes (8, 9). In addition to the double-stranded genome, 25 % of the RNA in purified reoviruses is short, single-stranded ribopolymers rich in adenine (17, 138). Their role is uncertain since they apparently are not required for infectivity (79). Attempts to demonstrate a linker function for these adenine-

6 VOL. 35, 1971 rich RNA species were unsuccessful (17, 138). It will be of interest to search further in reoviruses for an internal protein which is analogous to that found in influenza nucleocapsids. VIRUSES WITH SEGMENTED RNA GENOMES GENETICS The first demonstration of recombination by an animal virus was made with influenza virus (25, 26, 60). In this early work, the recombination frequencies observed were several-fold greater than the 1 % level expected for virions containing 2 x 106 to 3 x 106 daltons of RNA (44, 74). However, the frequencies were highly variable, since the characters studied were often polygenic and difficult to control experimentally. More recently, genetic studies have been done with conditional lethal, temperature-sensitive (ts) mutants isolated from stocks of influenza A strain WSN prepared with chemical mutagens. Simpson and Hirst (143) analyzed the progeny from chick embryo fibroblasts infected with different pairs of ts mutants. Certain pairs yielded recombinants with frequencies of 5 to 20%, close to the theoretical maximum of 25 % for independent assortment of unlinked markers; others resulted in no detectable recombinants. The results are consistent with the hypothesis that in infected cells genome fragments of both parental types are formed and then can undergo random assortment. This process apparently occurs during the early portion of the growth cycle (91). Mutant pairs which display a high frequency of recombination presumably are defective in different genome segments, whereas those which fail to recombine or do so at undetectable levels are mutated in the same segment. As suggested by Hirst (60), genetic interactions of influenza virus may be of two types: (i) exchange of entire segments resulting in high frequency recombination and (ii) crossing over between homologous genome segments yielding a low number of recombinants. The ts mutants can be grouped into at least five classes according to their ability to recombine. The classes may have their physiological basis in defects in each of the RNA fragments isolated from purified virions. Recombinants of avian and mammalian influenza A strains containing the neuraminidase from one parent and the hemagglutinin from the other have been obtained by using specific antisera as selective agents (162). The ease and consistency with which the stable antigenic hybrids were obtained suggest that the two antigens are coded for by different genome segments. The occurrence of such recombinants in nature might be important sources of new pandemic strains of influenza virus. Mackenzie has also obtained and studied a series of ts mutants of the WSN strain (91). They fall into five groups on the bais that mutants in 255 different groups recombine with frequencies greater than 1% and those within a single group recombine at frequencies less than 1 %. By adding receptor-destroying enzyme (RDE) after virus adsorption to synchronize replication and prevent adsorption of early progeny virus, the reproducibility of the recombination frequencies was markedly increased. The frequencies of recombination remained constant during the infectious cycle, indicating that a single cycle of genetic exchange occurred during the early portion of replication. Similarly, reovirus recombination is completed during the period of viral RNA synthesis 6 to 12 hr after infection (45). In addition to greater reproducibility, the use of RDE reduced the observed recombination frequencies of influenza virus by about twofold to a range of 1 to 8%. Mackenzie (91) has pointed out that his rlcombination data are consistent with a standard linear map for the influenza genome, in contrast to the interpretation of Simpson and Hirst (143). At least one mutant in each recombination group can be arranged on a linear map, and the distances between the mutants are additive. The map sequence obtained with pieces of chick allantoison-shell was verified in several other cell types. Ts mutants of reovirus type 3 have also been isolated (46, 68). Recombination results similar to those with influenza virus have been obtained for the reovirus mutants (45a). Five recombination groups were observed; mutants in different groups recombined with frequencies of 3 to 50%. If the lack of recombination among mutants in the same group is due to a defect in the same genome segment (as suggested for influenza virus), an additional five recombination groups may be anticipated, and three have been found recently (B. Fields, personal communication). Statistical analyses indicate that reovirus recombination occurs primarily by random assortment (45). Unlike influenza virus (91, 143), reovirus noninfectious particles probably do not participate in recombination since frequencies decrease when the multiplicity of infection is reduced from 10 to 1 PFU per cell (45). Preliminary biochemical and morphological studies of mutants selected from the five groups support the genetic classification (45b). At the nonpermissive temperature of 39 C, cells infected with group A mutants made normal amounts of viral RNA and whole virions were also observed. Group B mutants induced synthesis of lesser amounts of viral RNA and only after a delay;virus cores but not whole virus were formed. C and D mutants synthesized very little RNA and only the outer viral capsids were assembled. The single mutant in class E made the smallest amount of RNA, but, interestingly, viral proteins, sites of viral syn-

7 256 SHATKIN BACTERIOL. REV. thesis or "factories," and particles similar to wild-type virus were found in the cytoplasm of infected cells. Consistent with the high recombination frequencies observed with the ts mutants of reovirus and influenza virus, efficient recombination also occurs between active and UV light-inactivated virus (cross-reactivation; references 142, 154) and between two UV-inactivated strains (multiplicity reactivation; references 10, 94). Complementation has been reported with both virus types but not studied extensively (45a, 143). REPLICATION CYCLE Adsorption and Penetration Influenza virus adsorption is mediated by a specific interaction between receptor sites on the cell surface and the hemagglutinin spikes on the viral envelope (61, 125). The presence of cations is required for attachment, suggesting that electrostatic forces are important in the interaction. No specific mechanisms for reovirus adsorption have been documented. Glycoprotein interactions similar to those between cells and influenza may be involved, since reovirus hemagglutinating activity is inhibited by pretreating virus with N-acetyl-D-glucosamine (49). However, glycoproteins have not been reported in purified reoviruses, in contrast to the four found in influenza virus. The early events in the infectious cycles of reovirus and, to a limited extent, influenza virus have been studied by following the fate of purified, radioactive virus in infected cells (32, 34, 50). Although a useful approach, particularly in combination with electron microscopy, this method is subject to the important qualification that most of the radioactivity analyzed is associated with noninfectious particles since particle to PFU ratios of 10 to 100 are commonplace for many animal viruses. Penetration of reovirus into mouse L cells (140) and influenza virus into chick or monkey cells apparently is mediated by phagocytosis (33). In electron microscopy studies of infected cells, the input virus was observed in the cytoplasm enclosed within phagocytic vacuoles, and it was suggested that active engulfment or "viropexis" (43) occurs at the cell membrane. In influenza-infected cells, intact virus particles were not seen free in the cytoplasm, and the intravesicular particles were in various stages of degradation. Other studies with radioactive input virus also indicate that the envelope of influenza virus is degraded and the RNP or RNA is released during the first 1 to 2 hr of infection. The intracellular fate of the released influenza genome is not known further, but both the nucleus and cytoplasm are involved in the production of progeny virus (75, 114, 125). In the case of reovirus, the vesicle-enclosed parental particles are transferred to lysosomes by a process of membrane fusion. Within 1 hr after infection, 80% of parental, radioactive reovirus is found in the lysosome fraction (140). Digestion of the viral coat by preexisting cellular enzymes begins shortly after the parental virions appear in lysosomes, and by 3 hr after infection about 40% of the protein label is released as acid-soluble material. The majority of the input reovirus particles are converted by this degradative process to subviral particles which lack the outer capsomeric layer (147). The double-stranded genome is retained within the subviral particle, and no free double-stranded RNA has been detected in the cytoplasm of infected cells (51, 130). As a result of the loss of protein and concomitant retention of RNA, the subviral particles isolated at 3 hr after infection have a greater buoyant density in CsCl than the parental virions (130, 141). By 11 hr after infection, they return to a lower density, a process which is blocked by inhibiting protein synthesis with cycloheximide (141). This finding has been interpreted as evidence for the conservation of parental reovirus particles and their recoating by newly formed viral structural proteins. RNA Synthesis Reovirus single-stranded RNA. The intracellular conversion of reovirus to subviral particles described above can be simulated in vitro by treating purified virions with chymotrypsin or other proteolytic enzymes (137, 148). As in infected cells, the outer structural layer comprising four of the seven viral proteins is digested by chymotrypsin, and the proteolysis-resistant subviral particles remain (7, 146). They have an increased buoyant density in CsCl, g/cm3 versus for whole virions, and contain all 10 genome segments but not the adenine-rich, single-stranded ribopolymer. Treatment of most virus strains with chymotrypsin decreases their infectivity more than 1,000-fold (7, 145). However, strains have been isolated which remain infectious after proteolytic digestion and apparent conversion to subviral particles, indicating that the infectivity of reoviruses resides in the double-stranded RNA (148, 149). A highly active and stable RNA polymerase is firmly associated with the subviral particle prepared by chymotrypsin digestion (137). It is also manifest in virions after partial chymotryptic digestion or brief exposure to elevated temperatures (22). When subviral particles are incubated in vitro in a standard RNA polymerase assay mixture, synthesis of virus-specific, single-

8 VOL. 35, 1971 VIRUSES WITH SEGMENTED RNA GENOMES257 stranded RNA continues for several days. The enzyme is firmly bound to the template RNA and is not affected by actinomycin, rifampin, or a-amanitin. One strand of each of the 10 genome segments is transcribed by a conservative mechanism, and net quantities of RNA accumulate (7, 87, 145). Results of kinetic studies are consistent with the presence of one active, continuously reinitiating polymerase molecule per particle (7, 9, 145). This same activity is present in subviral particles isolated from the cytoplasm of cells early in infection, indicating that the polymerase is responsible for the synthesis of the initial viral mrna during replication (52). Synthesis of single-stranded viral mrna by the virion-associated polymerse probably begins in the cytoplasm within the 1st hr after infection, a time when partial proteolysis of parental virions occurs in the lysosomes (140). No evidence has been obtained for nuclear involvement in the production of reovirus-specific macromolecules in contrast to influenza-infected cells. The detection of virus-specific RNA is limited by the specific activity of the radioactive precursor used and the background of cellular RNA. In cells treated with 0.5,ug of actinomycin per ml, a concentration which selectively inhibits cell RNA synthesis (81, 133), reovirus-specific RNA is detected at 2 to 4 hr after infection, about 2 hr before the increase in infectious titer. When added at the time of infection, actinomycin at a concentration of 2 Alg/ml blocks reovirus RNA synthesis and virus replication (55). After RNA synthesis commences, it becomes less sensitive to actinomycin inhibition (81). Infection of cells with subviral particles prepared in vitro did not result in a shortened eclipse period (A. Shatkin, unpublished data), in agreement with the brief time interval necessary for the intracellular conversion of parental virions to particles with RNA polymerase activity. The rate of viral RNA formation increases after the 4th hr of reovirus infection. Presumably the single-stranded RNA formed by parental virions functions as a messenger for the production of new polymerase molecules. A large fraction of the single-stranded viral RNA present in infected cells is associated with polyribosomes which are actively synthesizing viral proteins (112, 136). In addition, single-stranded viral RNA produced in vitro by subviral particles has been shown to stimulate polypeptide synthesis by cell-free extracts of mouse L cells (86). The catalytic rise in viral RNA synthesis can be blocked by infecting cells in the presence of cycloheximide (160). Under these conditions, virus-specific RNA is made exclusively by the preformed polymerase of the parental virions. It includes transcripts from only 4 of the 10 genome segments (161), in contrast to the 10 singlestranded RNA species made in vitro by subviral particles prepared with chymotrypsin or isolated from infected cells. Intracellular transcription by the virion-associated polymerase is limited to one large, one medium, and two small genome segments. The same pattern was reported for viral RNA made during the early portion of a productive cycle of virus replication, 4 to 6 hr after infection (161). During the interval 6 to 8 hr after infection, a time when progeny virus is first present, transcription of all 10 segments occurs. The basis for the switch from early (limited) to late (complete) transcription remains to be determined, but the combined results of in vivo and in vitro studies suggest that restriction may be mediated by a preformed, possibly cellular, protein (A. Shatkin, unpublished data). In other similar studies, presumably with another variant of the Dearing strain of reovirus type 3, early mrna (2 to 4 hr after infection) included transcripts of all 10 genome segments (166). In this work, electropherograms of mrna hybridized to genome RNA were examined by autoradiography of intact polyacrylamide gels. Although 10 species were found and the pattern of transcription was the same between 2 hr after infection and 8 hr, a time when mrna synthesis was maximal, the frequencies of transcription of the L, M, and S segments differed. The relative amounts were in the order S > M > L, similar to that observed in vitro (7). The mechanism for control at the level of transcription is unknown. Reovirus genome RNA. Double-stranded viral RNA synthesis can be detected in reovirus type 3-infected mouse L cells at about the same time as the increase in viral mrna formation (48, 134, 135). Genome RNA comprises about onefourth of the total virus-directed RNA synthesized during replication. Its synthesis is concomitantly blocked when virus-directed protein synthesis is prevented by addition of cycloheximide (136, 160) or puromycin (82) at the time of infection. Double-stranded RNA formation, once initiated, is also dependent upon continued synthesis of protein. Addition of a protein inhibitor during an ongoing cycle of virus replication reversibly reduces double-stranded RNA production. The protein(s) involved in its synthesis apparently turns over rapidly. It is not known whether the necessary protein(s) is a completely new enzyme specified by the viral genome or a subunit which modifies the virion-associated polymerase or some other preexisting polymerase. An enzyme activity which catalyzes predominantly doublestranded RNA synthesis has been detected in a crude, large particle fraction isolated from in-

9 258 SHATKIN BACTERIOL. REV. fected cells (158). Further purification and study of this activity should provide a better understanding of double-stranded RNA replication. Although genome RNA formation is blocked by adding cycloheximide or puromycin during the replicative cycle, single-stranded reovirus RNA production continues. If the inhibitor is added during a period of rapid synthesis (9 to 11 hr after infection), single-stranded RNA accumulates at a slightly increased rate as compared to cells under normal conditions of infection. This observation is consistent with the recent report that at least a portion of the single-stranded viral RNA is a direct precursor of the double-stranded genome RNA (130). An earlier attempt failed to demonstrate a precursor relationship. Singlestranded viral RNA was specifically labeled with 3H-uridine during a "pulse" in the presence of cycloheximide (160). The inhibition of doublestranded RNA synthesis was then reversed by washing out the cycloheximide, and the fate of the 3H-labeled single-stranded RNA was followed. No 3H was detected in the newly formed genome RNA, indicating that the single-stranded RNA made in the presence of cycloheximide is not subsequently converted to duplexes. In a more recent study, a different approach was used to test the possibility that single-stranded viral RNA serves as a precursor of the genome RNA (130). Cells were labeled with 3H-uridine during different intervals after infection, and the double-stranded viral RNA was isolated. It was denatured and annealed with an excess of unlabeled single-stranded RNA made in vitro by the reovirus-associated polymerase. Since the in vitro product is copied exclusively from one strand of each genome segment, it will anneal to its complementary (minus) strand and displace the 3Hlabeled homologous (plus) strand from the duplexes in the annealed mixture. If the two strands of the double-stranded RNA are replicated simultaneously in infected cells, they will be equally labeled. Consequently, after annealing, approximately 50% of the label will be in duplexes and the remaining half, the plus strands, will be single-stranded and sensitive to ribonuclease digestion. Conversely, if there is asynchronous synthesis with the minus strands formed on templates of preformed plus strands, the bulk of the label will remain in duplexes after annealing. The results of the hybridization experiments indicate that, during a 30-min pulse with 3IHuridine (8 to 8.5 and 11 to 11.5 hr after infection), only minus strands in the double-stranded RNA are labeled. When the labeling period was increased to 8 or 11 hr, there was equal synthesis of plus and minus strands. Thus, it appears that in the infected cell the single-stranded viral RNA functions both as the messenger for viral protein synthesis and as a template for the formation of the minus strand of the genome RNA. Consistent with this possibility is the recent finding that the genome segments are perfect duplexes which are transcribed end-to-end (8). Whether different populations of plus strands are involved in each of these key functions remains to be determined. Influenza RNA. Several findings made in different laboratories suggest that influenza RNA replicates by a process similar to that of the simpler genomes of picornaviruses and RNA bacteriophages (19, 150). As in these systems, the single-stranded influenza RNA or RNP, after its intracytoplasmic release from parental virions, could serve as a messenger for the formation of a virus-specific, RNA-dependent RNA polymerase. The enzyme or modified forms of it would then replicate the input (plus strand) RNA via the formation of a complementary (minus strand) RNA. The presence of minus strands in influenzainfected cells has been reported (107), and doublestranded and multistranded viral RNA complexes have also been isolated (99). Kinetic studies with pulse-chase techniques suggest that the multistranded RNA is a replicative intermediate (102). In addition, an RNA-dependent RNA polymerase is induced after influenza virus infection of chick cells (62, 92, 119, 128, 144). This newly formed polymerase activity becomes detectable about 1 hr after infection and increases in parallel with viral RNA synthesis. However, minus strands which comprise about 10% of the singlestranded viral RNA have been found in cells within 15 min after infection, apparently before plus strands (129), suggesting the possibility that a virion-associated polymerase analogous to the reovirus enzyme may initiate influenza-specific RNA synthesis. An enzyme of this type has been uncovered recently in nine influenza strains including human, avian, and porcine types (28). As observed for other virus-associated polymerases including those of poxviruses (73, 98), reoviruses, VSV (6), NDV (66), and the RNA tumor viruses (5, 153), activity is apparent only after the virions are partially disrupted. Treatment of influenza with the detergents Nonidet P40 or Triton N101 is an effective means of uncovering the enzyme, but its RNA synthetic capacity is only 1 to 5% of that of the reovirus polymerase. Like reovirus, chymotryptic digestion of influenza converts virions to noninfectious cores by removing the outer envelope including the surface glycoproteins (131). Treatment of virions with the protease bromelain also digests three of the four viral glycoproteins and converts the spike-bound virions to smooth-surfaced, noninfectious par-

10 VOL. 35, 1971 VIRUSES WITH SEGMENTED RNA GENOMES 259 ticles which lack both NA and HA activities (30). The influenza cores prepared with chymotrypsin retained about one-half of the polymerase activity of detergent-treated virions, suggesting that the enzyme is internally situated rather than adsorbed to the particle envelope (28). In the presence of four ribonucleoside triphosphates and Mn2+, influenza virus activated by detergent treatment synthesizes RNA in vitro at a linear rate for at least 2 hr (28). The enzyme, like the RNA polymerase activity induced in influenza-infected cells, is unaffected by actinomycin (or rifampin) but is strongly inhibited by polyvinyl sulfate. Although not yet well characterized, the product after phenolization includes ribonuclease-sensitive minus strands and ribonuclease-resistant, and presumably doublestranded, RNA. The virion-associated and induced polymerases share a number of properties and may contain common polypeptides. However, the particle-associated enzyme is probably involved in the initiation of replication and not packaged fortuitously during virus maturation, since incomplete virions (von Magnus particles) which have a low specific infectivity are also deficient in RNA polymerase activity. Furthermore, genetic recombination can be demonstrated for the level of virus-associated polymerase activity, indicating that it is determined by the viral genome (28). After being synthesized by the parental virionassociated polymerase, the minus strand of influenza RNA and the reovirus single-stranded RNA could code for the formation of the virusinduced RNA polymerase activity in infected cells. Both plus and minus strands have been found in polyribosomes of influenza-infected cells (100), and reovirus single-stranded RNA (plus strand) which is present in polyribosomes is identical to that formed in vitro by the virionbound polymerase (85). The influenza RNA polymerase induced in infected cells apparently is virus-coded since it is specifically neutralized by homologous immune serum (124). It is found predominantly in the microsome fraction which on further purification reduces to an active 70S particle containing viral RNA and structural proteins (144). Some influenza RNA polymerase activity is also present in nuclei (128), and the virus-specific, nonstructural antigen observed in nucleoli of infected cells may be related to this nuclear activity (35). Whether influenza RNA formation is confined to the cytoplasm or nucleus is unclear at present (19). Pulse studies with radioactive uridine indicate that most of the viral RNA synthesis occurs in the cytoplasm. The protein of the RNP antigen is also synthesized in the cytoplasm and migrates to the nucleus, possibly together with the viral RNA (15, 24). The components of the RNA complex may be assembled into an antigenically active form in the nucleus under control of the nucleolar antigen. A low concentration of actinomycin added to cells at the time of infection blocks influenza RNA synthesis and virus multiplication (14). Specific inhibitors of deoxyribonucleic acid (DNA) synthesis do not affect influenza production (126). These findings have been interpreted to mean that a DNA-dependent step, i.e., a cellular function, is required for productive infection. The results of UV irradiation studies are also consistent with a requirement for cell mrna for the initiation of virus replication (11, 12). DNA-dependent RNA polymerase activity indistinguishable from that in uninfected cells increases by 60% at 2 hr after influenza infection of chick cells, but its role in the infectious cycle, if any, has not been shown (21). The induction of the viral RNA polymerase is prevented by actinomycin added during the initial stages of replication (62), and it was thought that this finding might explain the actinomycin sensitivity of influenza. Many investigators have reported that influenza replication is highly sensitive to actinomycin during the early phase of infection but becomes resistant as the cycle progresses. However, in a recent study, the addition of 1 jug of antibiotic per ml at any time during infection irreversibly reduced influenza virus RNA synthesis by 95% in chick cells (57). Viral protein synthesis continued for long periods, suggesting that influenza mrna is stable and continues to be translated in the presence of actinomycin (57, 116). A similar finding has been reported for actinomycin-treated, reovirus-infected cells (90a). Thus, although in vitro activity of the influenzainduced RNA polymerase is not affected by actinomycin, viral RNA synthesis in infected cells is apparently at least partially sensitive to the drug throughout infection. Furthermore, Scholtissek and Rott were able to isolate viral RNA polymerase activity from cells in which influenza RNA synthesis has been blocked by adding actinomycin at 2 hr after infection (129). It should be emphasized that the bulk of the single-stranded RNA formed in vivo corresponds to that present in virions (plus strands), whereas the in vitro product of Scholtissek was predominantly minus strand (122). Perhaps cell-coded proteins induced by infection act as modifiers of the influenza RNA polymerase(s) in a manner analogous to the host factors of Q3 bacteriophage RNA polymerase (150). The reports of influenza minus strands, plus strands, and double-stranded products obtained in different laboratories with partially purified enzyme preparations could result from

11 260 SHATKIN BACTERIOL. REV. the loss or inactivation of such factors. The appearance of the influenza-specific nucleolar antigen is also prevented by actinomycin. Since low levels of the drug are known to alter the structure of nucleoli, it has also been suggested that its inhibitory activity could be explained on this basis (35). The influenza-induced RNA polymerase has not been purified free from its template. Its reported RNA dependence is based on the inhibition of activity observed with ribonuclease but not deoxyribonuclease treatment. Although the general RNA polymerase properties such as triphosphate and cation requirements are similar for activities prepared in various laboratories, the products obtained have been different. Clearly, an understanding of the role of the induced polymerase in influenza RNA replication will require the isolation and study of a highly purified enzyme. Protein Synthesis Reovirus. The structural proteins found in purified reovirus constitute several biological functions which are also present in influenza virus, including hemagglutination, group and typespecific antigenicity, nucleoside phosphohydrolase, and RNA polymerase activity. The myxoviruses also contain neuraminidase which can be released from virions in an active form (55a, 83, 125). In contrast, the seven reovirus structural proteins have been separated only after denaturation. Consequently, the biological function of each polypeptide is unknown, and the synthesis of active viral proteins is difficult to follow in reovirus-infected cells. However, all seven structural polypeptides can be identified on the basis of molecular weight as early as 3 hr after reovirus infection by SDS-polyacrylamide gel analysis of cytoplasmic extracts (166). No evidence has been found for a nuclear phase in reovirus multiplication. Host protein synthesis continues at a reduced rate in reovirus-infected cells, but the background of newly formed cellular proteins can be diminished further by actinomycin treatment. As infection proceeds, the rate of viral protein formation increases to a maximum at about 9 to 10 hr, when it comprises more than 50% of the total nascent protein. The relative rates of formation of X, /5, and a proteins remain similar throughout infection; i.e., there are no distinct early and late protein classes. However, small shifts occur in the relative synthetic rates of proteins within a size class. In addition, the A, polypeptide is a precursor Of /2, and its earlier formation and cleavage to A2 result in an apparent change in their relative synthetic rates at early versus late times after infection (166). Cytoplasmic polysomes which have been shown to contain reovirus-specific, single-stranded RNA are probably the sites of synthesis of viral proteins (122, 136). Presumably the first viral polysomes contain mrna synthesized by the virion-associated RNA polymerase in both reovirus- and influenza-infected cells. The 10 single-stranded RNA species transcribed from the L, M, and S genome segments correspond in chain length to the values expected if one assumes that each functions as monocistronic reovirus messenger for the X,,u, and a polypeptides, respectively. Since there are 3 L, 3 M, and 4 S segments and 2 X, 2 At, and 3 a polypeptides, 7 of the 10 mrna species can be accounted for (166). If Iu is the primary gene product which is converted to /52, a maximum of six of the mrna species are translated to form viral structural proteins. It is not known whether infected cell polyribosomes separated according to size synthesize different classes of viral polypeptides and contain the corresponding species of mrna. Only the seven structural proteins have been detected in extracts of reovirus-infected cells, even under conditions of long pretreatment with low concentrations of actinomycin to reduce the background level of cellular proteins; however, all 10 mrna species are present in polyribasomes (R. Ward, A. K. Banerjee, and A. Shatkin, unpublished data). The three to four additional proteins which could be virus-coded may be present in infected cells at concentrations below the limits of resolution. Such a possibility implies that regulation occurs at the level of translation, since all 10 mrna species are readily discernible in infected cells (161, 166). A comparison of the relative numbers of mrna molecules transcribed from the different genome segments versus the number of corresponding polypeptide molecules formed indicates that, in addition to transcriptional control, the frequency of translation of the individual mrna species is regulated (166). For example, the M genome segments are transcribed less frequently than the S segments but apparently are translated more often since there are a greater number of,ll than a polypeptides formed during infection. The molecular basis for translational control may become clearer when the base sequences, including the location and number of initiating codons, of the reovirus mrna species are available. Influenza. Influenza infection leads to the synthesis of several major viral proteins in the cytoplasm of infected cells (31, 42, 152). Soon after infection, host protein synthesis decreases markedly, and by 1 to 1.5 hr viral protein formation predominates (42). The polypeptides which

12 VOL. 35, 1971 are incorporated into purified virions are synthesized concomitantly and at about the same relative rate throughout infection. Analysis of total protein synthesis in infected cells indicates that the polypeptide found in RNP is formed at slightly greater rates late in infection. In any case, as in reovirus-infected cells (166), there are no clearly discernible early and late classes of influenza structural proteins. The molecular weights of the major viral polypeptides vary somewhat when synthesized in different host cells (42). For the glycoproteins, the variation may be accounted for by host-dependent alterations in the carbohydrate portion of the molecule (30, 84, 131). The molecular weight differences would also be explained if the influenza structural polypeptides were derived from a larger precursor molecule by post-translational cleavage in a manner analogous to that demonstrated for enterovirus protein production. Peptide analyses of tryptic digests of influenza polypeptides have been presented in support of this possibility (42). A heterogeneous protein peak of molecular weight about 100,000 has been observed in cells at an early time after infection (42). However, an unequivocal, direct demonstration of precursor-product relationships for the viral polypeptides in infected cells, for example by pulse-chase experiments, remains to be done. In addition to the structural polypeptides, as many as five virus-induced antigens can be detected by immunodiffusion tests of infected cell lysates prepared 2 to 4 hr after infection. The function of these proteins is unknown, but one or more may correspond to the virus-induced RNAdependent RNA polymerase (35). Unlike reovirus, a nuclear phase is important for influenza multiplication. Autoradiographic and cell fractionation experiments indicate that the polypeptide moiety of the RNP migrates to the nucleus from its cytoplasmic site of synthesis, presumably the polyribosomes (15, 24, 152). It accumulates in the nucleoplasm (151), perhaps as a result of binding to RNA. Minus and plus strands of viral RNA bind to a similar extent to RNP-antigen protein in vitro (123). Minus strands also comprise a small fraction (less than 10%) of the RNA in RNP extracted from infected cells (107). Since only plus strands are assembled into virions, some mechanism must operate to select viral genomes at the RNP level. At later stages in the influenza cycle, RNP is also present in the cytoplasm (24). It is not known whether it has been assembled in the nucleus and subsequently released into the cytoplasm or whether both synthesis and assembly of RNP can occur in the cytoplasm during the later phases of multiplication. In view of the presence of plus RNP in VIRUSES WITH SEGMENTED RNA GENOMES 261 virions, it would be interesting to compare the strandedness of nuclear versus cytoplasmic RNP. Perhaps only the RNP containing plus strands appears in the cytoplasm. Consequently, assembly of virions which takes place at the cell surface would be limited to plus strands of RNA (152). A second viral structural protein of molecular weight about 20,000 also migrates to the nucleus after synthesis in the cytoplasm of infected cells. It accumulates almost exclusively in the nucleoli. Inhibition of protein or RNA synthesis by cycloheximide or actinomycin does not prevent the migration process (151). Metabolic poisons including sodium fluoride, potassium cyanide, and 2,4-dinitrophenol are also noninhibitory, suggesting that the newly formed viral proteins are not transported by an energydependent process but diffuse to the nucleus. Another major polypeptide present in influenza virions, the HA, is found only in the cytoplasm of infected cells. It is incorporated into the host cell membrane at discrete sites where mature virions are assembled and released by a budding process (37). Host membrane proteins are not present in mature virions, indicating that they are displaced by viral polypeptides at the budding sites (63). Although the polypeptides are viruscoded, the carbohydrate portion of the structural glycoproteins is apparently determined by the host cell (84). The lipids of the viral envelope are also derived from host components (20, 72, 78). Maturation Reovirus multiplication is confined to discrete regions in the cytoplasm of infected cells (140). Presumably the following series of complex events leading to assembly of progeny virions occurs entirely within these developmental foci. The parental particles are first converted to subviral particles which are conserved at this stage of disassembly. The duplex segments are then continuously transcribed in situ by the particle-associated RNA polymerase (transcriptase). A portion of the single-stranded product binds to neighboring ribosomes and is translated. One or more of the resulting proteins may be the enzyme(s) which utilizes another portion of the single-stranded product as a template to complete the formation of double-stranded progeny genomes. The latter process may occur in association with subviral particles under conditions in which the genome segments and their singlestranded products are linked. The enzyme (replicase) which makes double-stranded RNA could consist of two (or more) polypeptides, one identical to the transcriptase and a second which modifies the complex to permit utilization of a

13 262 SHATKIN single-stranded template for double-stranded RNA synthesis. The modifier subunit might be released upon completion of the double-stranded RNA, leaving the template-bound transcriptase ready to begin the cycle again. Double-stranded reovirus RNA is not found free in the cytoplasm of infected cells and is probably enclosed within subviral particles during, or shortly after, synthesis. At some stage between 6 and 12 hr after infection, however, the genome segments are available for genetic exchange; perhaps this process takes place at the level of single-stranded RNA transcripts of the duplex segments. The subviral particles are then converted to virions by the addition of structural proteins. Most of the progeny infectious particles remain cell-associated. The close coordination between viral RNA and protein synthesis probably facilitatesthe availability of complete genomes for assembly into virions and obviates the necessity for correctly linking 10 independent molecules of doublestranded RNA. The influenza parental virions are also degraded in the cytoplasm of infected cells. This process may occur in stages, the first resulting in the removal of the viral envelope and the formation of glycoprotein-deficient cores. RNA polymerase in the cores could then produce RNA strands complementary to the viral genome. Further degradation of parental particles would lead to release of viral RNP or RNA. Thus, both minus and plus strands would be available to function as messengers for the cytoplasmic synthesis of virus-specific proteins including the RNA-dependent RNA polymerase. In the infected cell, this enzyme presumably uses the minus RNA (or RNP) as template for the replication of viral genome or plus RNA. Recombination, whether by exchange of RNA segments or crossing over, apparently occurs during this portion of the cycle. Among the viral structural proteins, an argininerich polypeptide binds strongly to the newly formed viral RNA, and the ribonucleoprotein complex appears in the nuclei during the earlier stages of the infectious cycle. At later times, the cytoplasm also contains viral RNP. The viral hemagglutinin remains in the cytoplasm and, together with the neuraminidase, is incorporated into localized sites in the host membrane by displacement of the cell membrane polypeptides. Addition of the carbohydrate moiety of the viral glycoproteins may occur at this time in the maturation process. The membrane forms evaginations or buds at the sites of viral protein incorporation. RNP is enclosed within the buds which pinch off by membrane fusion at their base releasing mature virions. Viral neuraminidase is probably BACrERIOL. REV. involved in the release of virus from cell surface receptors, a process which is inhibited by antibody to the enzyme (29, 132). CONCLUSIONS From the combined biochemical and morphological results, it appears that both single-stranded (influenza) and double-stranded (reovirus) RNA viruses may contain segmented genomes. The presence of 5-terminal nucleoside di- or triphosphates indicates that the segments are individually synthesized and subsequently assembled to form the viral genome. The mechanism for ordering and linking these subunits remains an important and interesting problem which may also provide a useful biological system for studying nucleic acid-protein interactions. Synthesis of the various viral RNA species in infected cells is mediated by newly induced and parental virion-associated RNA polymerases. The reovirus-associated RNA polymerase is highly active in vitro, continuously reinitiating and terminating synthesis of large quantities of distinct species of viral mrna. The size of the mrna species is consistent with their functioning as monocistronic messengers for the several viral structural proteins which have been characterized. Thus, this system is potentially a unique one for studying mammalian cell-free transcription and translation. Information on the control of transcription and translation, as well as genetic interactions of the subunits, should be relevant for understanding similar processes in higher organisms. Finally, the maturation of influenza includes the packaging of the ribonucleoprotein segments within a lipoprotein envelope consisting of virus-specified proteins and cellular lipids. Studies of myxovirus assembly may be helpful in learning more about the biosynthesis and structure of biological membranes. ACKNOWLEDGMENT I am grateful to P. H. Duesberg, R. 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