Minireview Vol. 268, No. 6, Issue of February 25, pp. 3797-3800,1993 n l e JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U S A .

Double-stranded RNA Virus Replication and Packaging

Reed B. WicknerS From the Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Marylnnd 20892

Double-stranded RNA viruses are known in all major groups of organisms, from bacteria and fungi to animals and plants. The first demonstration of dsRNA' in nature was that in reoviruses by Gamatos and Tamm (l).' In addition to the Reoviridae (including reoviruses and rotaviruses) having 10- 12 dsRNA genomic segments, the two-segmented Birnaviri- dae (infectious bursal disease virus of chickens, pancreatic necrosis virus of fish), the three-segmented phage P6, and the unsegmented Totiviridae (such as the L-A virus of yeast and other fungal and parasite viruses) have dsRNA genomes. Rotaviruses are a major cause of death from diarrhea in man, and other members of the Reoviridae are important causes of morbidity and mortality of many farm animals and crops. The antiviral activity in mice of Penicillium extracts was shown in the 60s to be due to induction of interferon by dsRNA from intracellular virus.

While the overall replication cycles for dsRNA viruses were clear almost 20 years ago, only recently with the development of molecular cloning techniques and in vitro transcription, replication, and packaging systems have detailed mechanisms for these processes come hazily into view. This review will emphasize these recent developments and particularly the yeast L-A system in which these aspects, as well as the study of the roles of chromosomal genes in viral propagation, are especially well developed.

Recent reviews of the Reoviridae (2-41, bacteriophage P6 (5), the L-A virus of Saccharomyces cereuisiae (6), dsRNA viruses of protozoa (7), and dsRNA viruses of lower eukar- yotes in general (8) have appeared.

Viral Replication Cycles &RNA replication occurs in the cytoplasm for all dsRNA

viruses that have been investigated. Transcription, defined as the synthesis of viral (+)-strands from a dsRNA template, takes place within viral particles or core particles. One inter- esting exception is the dsRNA replicons that mitigate the virulence of the chestnut blight fungus, Cryphonectria paras- itic~. These are found in membranous vesicles and lack a substantial protein coat (9).

The new (+)-strands are generally extruded from the viral particles. These (+)-strands are then translated to make viral proteins. It is then the same (+)-strands that are packaged to make new particles or subviral particles. Once the new parti- cles or cores have formed, (-)-strand synthesis on the (+)- strand template (replication) completes the formation of new

$ To whom correspondence should be addressed Bldg. 8, Rm. 207, NIH, Bethesda, h4D 20892. Tel.: 301-496-3452; Fax: 301-402-0240.

'The abbreviations used are: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; ORF, open reading frame.

Because of space limitations, citations of original papers are largely limited to those not referenced in the reviews cited (Refs. 2- 8).

Pqno-Toxin

Head is full so new (+)strand is pushed out

slays Inside viral particle

FIG. 1. Viral replication cycles of the L-A virus and its satellite dsRNAs, M (1.8 kilobases encoding the killer toxin and immunity to the toxin) and X (a deletion mutant of L-A 630 base pairs in length). Intracellular particles, with an L-A- encoded coat, are found containing a single L-A &RNA molecule, either one or two M1 dsRNA molecules, or one to eight X dsRNA molecules. Particles containing more than one type of &RNA are not found in this system.

dsRNA. In systems where the virus is destined for export, addition of new layers of protein and/or membrane completes the virus reproduction cycle.

L-A is a 4.6-kilobase dsRNA virus that encodes its major coat protein (Gag) and its RNA polymerase, made as a Gag- Pol fusion protein by ribosomal frameshifting. L-A coat pro- teins can separately encapsidate and replicate the "satellite" dsRNAs, M (several are known: M1, Mz, . . .), encoding a secreted protein toxin (killer toxin, reviewed in Ref. IO), deletion mutants of M1 (called S mutants), or of L-A itself (one of which is called X dsRNA). The cycles for the L-A virus of S. cereuisiae, and its satellite dsRNAs, are shown in Fig. 1, as these are the focus of this review. Host cell (chro- mosomal) KEX genes are needed for killer g ress ion , MAK genes for viral propagation (maintenance of killer), and SKI genes for repressing viral propagation (mutants are super@- ers).

In Vitro Systems Since all known dsRNA virus particles include a viral RNA-

dependent RNA polymerase, viral RNA synthesis is easily observed in purified or even relatively crude virus prepara- tions. These activities are primarily (+)-strand synthesis on the viral dsRNA template if mature viral particles are used. Newly formed particles, containing only (+)-strands, can be isolated and carry out (-)-strand synthesis. Because neither the template nor the enzyme can be altered in these reactions, they are of use mainly to study the products of the reactions and the requirements for and effects of small molecules. The finding that L-A viral particles exposed to low salt conditions lose their dsRNA but can now specifically bind and replicate added viral (+)-strands or suitable artificial RNAs, and can transcribe added viral dsRNA, has allowed study of a number

3797

3798 Minireview: dsRNA Replication and Packaging

of these processes in detail (1 1). We refer to these as “opened empty particles.” Likewise, P6 provirus particles, analogous to reovirus cores, can package phage (+)-ssRNAs and convert them to the dsRNA form in uitro (12). Rotavirus subviral particles combined with a rabbit reticulocyte protein synthe- sizing system can carry out the replication and partial pack- aging of exogenous viral (+)-strands (13). These template- dependent systems all have the advantage that enzyme-tem- plate specificity can be examined.

Mechanisms Transcription ( = (+)-Strand Synthesis)-Different seg-

ments of the multisegmented dsRNA viruses share a common sequence at the 5’ ends of their (+)-strands, the starting point for transcription and therefore a presumed recognition site. The template-dependent in vitro transcription system de- scribed for L-A should provide a method to directly identify signals recognized by the transcriptase, but this remains to be done. The L-A transcription reaction requires an extract of (uninfected) host cells and a high concentration of polyeth- ylene glycol. Transcription is conservative in mammalian reo- and rotaviruses and in the L-A virus of yeast but semiconser- vative in the bacteriophage 46 and the Aspergillus (14) and Penicillium (14) viruses. The difference is only whether the template (-)-strand remains annealed to the new (+)-strand (semiconservative) or repairs with the parental (+)-strand (conservative).

Extrusion of Transcripts and the Headful Replication Model-New L-A or reovirus transcripts are all extruded from the particles. Is this an active transport process driven by an RNA exportase? Such a possibility is suggested for rotavirus by their requirement for a hydrolyzable form of ATP for transcription (15). The satellite dsRNAs of L-A have genomes ranging from one-eighth to one-half the size of L-A itself. Particles that are full, having about 1 L-A eq of dsRNA, extrude all their new (+)-strands from the particle, while those that are not often retain the new (+)-strand and convert it to a second dsRNA molecule within the same particle as its parent. This can continue until there are as many as eight dsRNA molecules in one particle in the case of the small satellites of L-A that are about one-eighth the size of L-A. This suggests that extrusion of transcripts is simply a conse- quence of the heads being full and is driven by the energy of polymerization, not by a specific exportase. This is referred to as “headful replication” and probably applies to a number of single-segment fungal dsRNA viruses (Totiuiruses) but perhaps not to the multisegmented dsRNA viruses (see “As- sembly and Packaging” below).

Translation of (+)-Strands-The (+)-strand transcripts ex- truded from dsRNA viral particles serve as mRNA to make viral particle proteins. The (+)-strands of L-A have two open reading frames, the 5’ gag ORF (680 residues) and the 3‘ pol ORF (868 residues), that overlap by 130 base pairs (Fig. 2). The gag ORF encodes the major coat protein, while pol has the consensus amino acid sequence patterns diagnostic of RNA-dependent RNA polymerases of (+)-ss- and dsRNA viruses (Ref. 16, Fig. 2). The pol ORF is expressed only as a Gag-Pol fusion protein (17) formed by a -1 ribosomal frame- shift event (16, 18), like that used by retroviruses ((19) re- viewed in Ref. 20). The frameshift event requires a slippery site on the mRNA of the form X XXY YYZ (where the gag frame is shown) (Fig. 3). This sequence allows the tRNAs at the A-site and P-site on the ribosome to both slip back one base on the mRNA and still have their non-wobble bases correctly paired. They do this because of the presence of an RNA pseudoknot just 3’ to this site (18), which arrests the ribosome’s progress (21).

-1 R~bosornal frameshift site,

L-A (t) strand * Packaging ion slgnd ._

5’ . qaq ORF I. nnl ORF

. . gagPo, fuwn protein

FIG. 2. Information encoded in the 4579-nucleotide L-A (+)-strand. The locations of RNA sites (for packaging, replication, and ribosomal frameshifting) and of encoded proteins (gag, major coat protein; pol, RNA-dependent RNA polymerase with ssRNA binding activity and encapsidation function) are shown.

..&

MI(+)- 5’ 3’

FIG. 3. Detailed structure of RNA sites on L-A (+)-strands that direct -1 ribosomal frameshifting ( top) and the packag- ing and replication of L-A and MI (+)+sRNA (middle and bottom). The -1 ribosomal frameshift event occurs at the slippery site and requires as well the adjacent RNA pseudoknot (see text). The (-)-strand synthesis reaction requires both the packaging site (for polymerase binding to template) and the 3’ end site at which polymerization of (-)-strands begins. The L-A and MI packaging sites include direct repeats (bored) whose role is unknown.

The efficiency of ribosomal frameshifting is critical for viral propagation (22). Normally, 1.8% of ribosomes frameshift at this site, and since there are about 120 Gag molecules per particle, this suggests there are 2 fusion protein molecules per particle, consistent with the observed ratio of Gag-Pol to Gag proteins in whole cell extracts or isolated viral particles. A deviation of more than 2-fold from this normal ratio results in loss of viral propagation. Even changes within this range result in decreased copy number of the viral genome. AS discussed below, this altered ratio of Gag-Pol to Gag must have its adverse effect via the assembly and packaging process.

Although ribosomal frameshifting is not widely used in dsRNA viruses, its discovery in L-A did lead to a general idea about the use of splicing, termination read-through, RNA

Minireview: dsRNA Replication and Packaging 3799

editing, and frameshifting by certain classes of viruses (16). Double-stranded RNA viruses, (+)-strand RNA viruses, and retroviruses all use their (+)-strands for three functions: as mRNA, as the species packaged to make new virions, and as a template for replication. If they alter those (+)-strands for translation purposes, by splicing or editing it, they will be creating mutant viruses unless, in making the alteration, they remove a site necessary for packaging or for replication. Presumably for this reason, dsRNA and (+)-sRNA viruses do not splice or edit their (+)-strands, and retroviruses remove the packaging site when they splice. Both ribosomal frame- shifting and the read-through of termination codons can be used to make the Gag-Pol fusion proteins without altering the mRNA.

Reovirus also uses translational tricks, producing two pro- teins from the S1 segment, a l and als. a l is an outer capsid protein that determines tissue tropism and many pathological features of viral infection. als is a non-structural protein of unknown function (23-25). These proteins are encoded in different reading frames, with u l starting at the first AUG (base 13) and als starting at the second AUG (base 71). The recently described monosegmented dsRNA virus LRVl of Leishmania has a replication cycle similar to that of L-A (26) and, judging by the sequence of the genome, may make a Gag- Pol fusion protein by a +1 frameshifting event (27).

Assembly and Packaging-The viral (+)-strand is the spe- cies packaged to form new viral particles in all dsRNA viruses studied. Reovirus assembly and packaging are thought to occur in cytoplasmic “factories,” inclusion bodies dense in virus proteins.

In the case of L-A, opened empty particles were found to bind specifically either L-A or MI viral (+)-strands. The site bound (Fig. 3) is about 400 nucleotides from the 3’ end of the L-A (+)-strand, and a similar site was found similarly located on the MI (+)-strand. Either site was sufficient to produce packaging of an unrelated transcript in vivo, indicating that these are the viral packaging sites (28). The stem structure, the sequence of the loop, and the A residue protruding on the 5‘ side of the stem are all necessary for activity.

Expression of gag alone is sufficient to form morphologi- cally normal (albeit empty) virus particles, indicating that the information for gross particle structure is independent of pol. However, the packaging of RNA having the viral packaging site requires the additional expression of the N-terminal fourth of the pol ORF (28). The Pol domain of the Gag-Pol fusion protein has s R N A binding activity, and this suggests that Pol actually binds to the packaging site on the RNA. The Gag domain of the fusion protein presumably polymerizes with free Gag molecules to produce complete particles, each with a single viral (+)-strand enclosed (Fig. 4). The host factor needed for the in vitro (+)- and (-)-strand synthesis steps (see below) is presumably also packaged at this point.

That the ratio of Gag-Pol fusion protein to Gag protein is critical for viral propagation can be interpreted in terms of this model. If the Gag-Pol fusion protein is in excess, many more particles would be started, but there would not be sufficient Gag to complete them. A relative excess of Gag protein might result in the particles being closed before the pol domain had a chance to find a (+)-strand to package.

One of the classical problems in RNA virology has been the mechanism by which viruses with multiple segments manage to package one of each in each particle. This question applies to the Reoviridae, the Orthomyxoviridae (such as influenza), the Bunyaviruses, and Arenaviruses. It has been suggested that the mechanism might involve complementarity between regions of the segments, but this has not been found. The dsRNA bacteriophage P6 has 3 segments, and an in vitro system capable of packaging viral (+)-ssRNA, replicating it

(+) ssRNA Genomic

& 4 .

gag-pol fusion protein

? ssRNA binding (polymerase) domain

&coat Drotein domain

I c“

+ fusion protein primes capsid

formation FIG. 4. Packaging and assembly model. The Pol domain of

the Gag-Pol fusion protein (perhaps as a dimer) binds specifically to the RNA packaging site on the viral (+)-strands, and the Gag domain associates with free Gag protein to prime polymerization of the coat. This results in the encapsidation of a single viral (+)-strand, which is then converted within the particle to dsRNA form. This model is supported by the existence of a Gag-Pol fusion protein, by the ssRNA binding activity of the Pol domain, and by the requirement for the N-terminal fourth of pol for packaging (but not for assembly).

to dsRNA, and forming infectious particles has been devel- oped (12). The packaging and (-)-strand synthesis is carried out by the isolated viral procapsid, a core structure comprising four of the viral proteins. Using this system, it has been found that (-)-strand synthesis on (+)-strand templates packaged in vitro depends on all three (+)-strands having been packaged (29, 30). No single segment or pair of segments is sufficient for synthesis to proceed. Only when all three have been packaged are (-)-strands made. Moreover, fragments of the full-length (+)-strands can be packaged, and the RNA pack- aging sites have been localized to their 5’ ends. The data indicates that the procapsids (cores) have specific binding sites for each segment (+)-strand. However, the answer may differ in some multisegmented animal viruses, whose low ratio of infectious to non-infectious particles and other data (31) suggests that particles may not precisely package one of each segment, as does (06.

The (06 system also shows that dsRNA virus assembly can proceed by formation of a structure followed by packaging, like many DNA phage, rather than the more concerted mech- anism postulated for L-A, in which the structure is thought to form around the RNA.

Minus Strand Synthesis-L-A’s (-)-strand synthesis step has been studied in detail using the opened empty particles described above as the enzyme source and natural viral (+)- strands or synthetic RNAs as templates (11,32). The reaction is specific for viral (+)-strands and requires an as yet unchar- acterized “host factor,” present in extracts of cells lacking L- A. The RNA signals required for template activity include the 3”terminal region of the L-A (+)-strand and an internal site 400 nucleotides from the 3’ end that largely overlaps with and may be identical to the packaging site (Fig. 3). Within the essential 3’-terminal region of L-A, the 3‘ end 4 bases are essential, as is an immediately adjacent stem-loop. The se- quence of the stem is not important, only that it be a stem, but the sequence of the loop is important. Remarkably, the 3‘ end of MI that differs in sequence and structure from that of L-A can substitute for L-A’s 3‘ end.

The RNA polymerase probably binds first at the internal site, then loops around to find the 3’ end at which synthesis begins. I t does not need to track along the RNA to find the 3‘ end nor does the internal site stimulate polymerase activity by an allosteric effect.

Rotavirus-infected cells will amplify and express a gene 9

3800 Minireview: dsRNA Replication and Packaging

(+)-strand analog in which the chloramphenicol acetyltrans- ferase gene is flanked by the 5’ and 3‘ non-coding RNA of gene 9 (33). Using this system, the 3”terminal 19 nucleotides and 5’-terminal44 nucleotides were found to be sufficient to promote amplification of transfected RNA (33). This suggests that rotavirus does not have an internal site such as that described above for L-A and MI.

Role o f Host Genes in Viral Propagation The highly developed classical genetics and molecular tech-

niques in the yeast system have facilitated the study of the role of host components in viral propagation and in the expression of viral-encoded information. For example, the KEXl and KEX2 genes initially identified as required for killer expression (secretion of active killer toxin encoded by the M dsRNA satellite of L-A) and for a-pheromone secretion and mating by a strains (34) were found to encode the proteases responsible for the processing of the prepro-toxin and pro-a-pheromone (10, 35). These proteases recognize dibasic cutting sites, like those required for processing insulin, prepro-opiomelanocortin, and other mammalian prohor- mones (35). This led to the identification of several mamma- lian enzymes responsible for prohormone processing, an area recently reviewed by Steiner et al. (36).

The acetylation of the N terminus of the L-A Gag protein is catalyzed by the host MAK3 protein, an N-acetyltransferase (37). This acetylation is necessary for viral particle assembly. Rous sarcoma virus and a number of plant RNA viruses have major coat proteins that are N-terminally acetylated. Simi- larly, the reovirus structural protein p1 is N-myristylated (38), and this modification is necessary for cleavage of p1 to form the mature form, p1C (39).

The formation of the gag-pol fusion protein involves ribo- somal frameshifting, and the efficiency of this process is critical to viral propagation as discussed above. Host mutants with altered efficiency of ribosomal frameshifting are defec- tive in viral p r~pagat ion .~

The SKI products repress replication of the L-A, M, and L-BC dsRNA replicons as well as of 20 S RNA, an ssRNA replicon, and this is their only essential function for the cell. Their mechanism of action is not yet clear, but Widner and Wickner4 argue that they may specifically block translation of viral RNAs.

The “host factor” required for in vitro (+)- and (-)-strand synthesis has been discussed above and remains to be char- acterized.

Questions for Future Work The tools available for the yeast L-A virus have made it an

attractive object for study, but an understanding of the proc- esses outlined here, for L-A and for other dsRNA viruses, remains for future work.

What is the host factor(s) necessary in the RNA synthesis steps? How do the SKI and MAK genes work for the host and virus? What are the functions of the many additional proteins encoded by Reoviridae? Do they serve functions that the host cell performs for L-A or are they indicative of more complex pathways? What is the signal for transcriptase?

J. D. Dinman and R. B. Wickner, unpublished data. W. R. Widner and R. B. Wickner, unpublished data.

How does L-A get its (+)-strand translated without a cap (reo- and rotaviruses have caps)? Can L-A be launched from a cDNA clone or transfected as RNA, an especially important issue for use of this system in biotechnology? What is the structure of L-A and other dsRNA viruses? L- A is much smaller than the Reoviridae, and its structure may be similar to that of reovirus cores. Can RNA-dependent RNA polymerase be prepared in sol- uble active form for studies of its enzymology and structure? How does packaging actually work? How are all segments recognized? The dsRNA viruses have many common features. Differ-

ences among free-living organisms and parasites are generally interpreted in terms of their adaptive value in occupying some ecological niche. Likewise, differences in replicative strategy between dsRNA viruses and (+)- or (-)-ssRNA viruses may have some as yet unrecognized adaptive significance.

Acknowledgment--I thank Peter Collins for critical reading of the manuscript.

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