key: cord-0861737-ck7eto16
authors: Baric, Ralph S.; Shieh, Chien-Kou; Stohlman, Stephen A.; Lai, Michael M.C.
title: Analysis of intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription
date: 1987-02-28
journal: Virology
DOI: 10.1016/0042-6822(87)90414-4
sha: 771006c821f2bad4ea3eb592173208e7fb51834e
doc_id: 861737
cord_uid: ck7eto16

Abstract We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived. The size of each subset correlates with regions of secondary structure, whose free energy ranges from −1.6 to −77.1 kcal/mol, in each of the mRNAs examined. In addition, identical subsets were detected on the replicative intermediate (RI) RNA, suggesting that they represent functional transcriptional intermediates. The biological significance of these small RNAs is further supported by the detection of leader-containing RNAs of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two MHV recombinant viruses. These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription.

We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived.

The size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77. 

Mouse hepatitis virus (MHV), a member of Coronaviridae, contains a linear single-stranded and positivesense RNA of 5.4 X 1 O6 Da (Lai and Stohlman, 1978) . The genomic RNA is enclosed in a helical nucleocapsid structure constructed from multiple copies of nucleocapsid protein (N) (Sturman, 1977) . Virions are enveloped and contain two virus-specific glycoproteins of 180 kDa (E2) and 23 kDa (El) (Sturman et al., 1980) . Upon entry into the host cell, the genomic RNA is translated into an early polymerase which directs the synthesis of a full-length negative-sense viral RNA (Brayton et a/., 1982; Lai et al., 1982) . In turn, the negative-sense RNA is transcribed by a late or altered early polymerase into the genomic RNA and six subgenomic mRNAs (Bray-ton eta/., 1984) . These mRNAs range from 0.6 to 5.4 X lo6 Da in length and are arranged in the form of a nested set from the 3'-end of the genome (Lai et a/., 1981) . The 5'-ends of each mRNA and the genomic RNA appear to contain an identical leader sequence of about 72 nucleotides, which are encoded only at the Y-end of the genome (Lai eta/., 1983 (Lai eta/., , 1984 Spaan et al., 1983) . The uv transcriptional mapping studies suggest that the mRNAs are not derived from cleavage of a large precursor RNA (Jacobs era/., 1981) nor is there any evidence indicating that nuclear factors are required for MHV replication (Brayton et a/., 1981; Wilhelmsen et al., 1981) . These data suggest that conventional eukaryotic RNA splicing is not involved in MHV transcription. Analysis of MHV replicative intermediate and replicative form RNAs suggests that a free leader RNA may be involved in subgenomic mRNA synthesis (Baric eta/,, 1983 ). In addition, discrete small leader-related RNAs of various sizes have been detected in MHV-infected cells (Baric et a/., 1985) . We have also isolated a ts mutant of MHV which synthesizes only small leader RNAs, but not mRNAs at the nonpermissive temperature (Baric et a/., 1985) . These data suggest that the MHV mRNAs are synthesized discontinuously and may utilize a single or multiple free leader RNAs as primers for the transcription of different subgenomic mRNAs. Additional proof that free transacting leader RNA(s) function in MHV transcription came from the demonstration of reassortment of leader RNAs between two different strains of MHV (Makino et a/., 1986b) . However, the exact leader RNA species which function in MHV transcription has not been identified.

RNA recombination occurs at very high frequencies during mixed infection with two heterologous strains of MHV (Lai et a/., 1985; Makino et a/., 1986a) . These data, coupled with the presence of discrete large leader-containing RNAs which range from 84 to 1000 nucleotides in length in MHV-infected cells (Baric et a/., 1985) suggest that discontinuous RNA intermediates may be dissociated and reassert between viral RNA templates to generate recombinant viruses by a copy-choice mechanism (Makino eta/., 1986a). Therefore, the larger leader-containing RNAs present in MHVinfected cells may represent functional intermediates of RNA transcription and recombination. In this paper, we have analyzed the origin, structure, and probable mechanism of synthesis of these RNAs. The data suggest that MHV RNA transcription may pause at sites corresponding to hairpin loops in the RNA template, and they support a mechanism of discontinuous RNA transcription in which RNA intermediates can be dissociated and reassociated with the RNA template intermittently during the course of transcription.

The A59 strain of MHV was propagated in either DBT or L2 cells at 37" in Dulbecco's modified MEM medium supplemented with 10% fetal calf serum containing 100 pg/ml penicillin and 50 pg/ml streptomycin. Infection was performed at a m.o.i. of 1 to 5, as previously described (Baric er a/., 1983).

Preparation of MHV intracellular RNA RNA was extracted from infected cells by the phenol/ chloroform method between 5 and 7 hr postinfection (70% CPE) (Baric et al., 1983) . Following ethanol precipitation, the RNA was washed once in 70% ethanol to remove excess salts and analyzed by electrophoresis on polyacrylamide gels as previously described (Baric et al., 1985) .

Intracellular RNA was extracted from infected cells as described above and applied to 15-30% sucrose gradients made in NTE buffer (0.1 ILI NaCI, 0.01 M Tris-HCI, pH 7.4, and 0.001 1\/1 EDTA) containing 0.1% SDS. RNA was sedimented at 45,000 rpm in an SW55Ti rotor for 2 hr at 18" and 0.2-ml fractions were collected. In duplicate gradients, 28, 18, and 4 S RNA were included as size markers. To isolate MHV RI RNAs, the 18-40 S RNA fractions were pooled and precipitated with ethanol. It has previously been shown that the RI RNAs are contained within this size fraction (Bark et al., 1983 (Bark et al., , 1985 Sawicki and Sawicki, 1986) . The 4-10 S RNA fractions were collected as the free small RNAs.

The cDNA clones of MHV genomic RNA used in this study are summarized in Fig. 1 . Clones F82 and C96 represent overlapping regions in the 5'-end of the genomic RNA (Shieh et a/., 1987). F82 is a 3.8-kb clone representing the very 5'end of the genome including the leader RNA sequences, while clone C96 represents internal sequences in gene A. Two other clones representing the internal sequences of MHV genome were also used: Clone D63 contains entire sequences of genes D and E and part of genes C and F (Shieh, unpublished), and clone PHN42 spans the entire gene F (kindly provided by Dr. Heiner Niemann, Giessen, Germany). An internal Xbal fragment (0.4 kb), designated D63X, of clone D63, was used as the probe for gene D. This fragment includes nucleotides 47-375 in the mRNA 4 coding sequence as reported (Skinner and Siddell, 1985) . The construction of subclones derived from PHN 42 followed the procedures described by Maniatis er al. (1982) . Subclone El-500 was derived by blunt-end ligating a 509 nucleotide Ddel fragment, located between nucleotides 90 and 609 in the gene F sequence (Armstrong et al., 1984) into plasmid pT7-2 at the Smal site (pT7-2 was purchased from Amersham). Subclone El-300 was derived from a 246-nucleotide fragment, located between nucleotides 534 and 780 in the El RNA (Armstrong et a/., 1984) and was inserted into plasmid pT7-2 at the /-/indllllSmal site. El-L was excised from PHN 42 by an EcoRIIScal restriction digestion. It contains the first 84 nucleotides of the mRNA 6, including the 72-nucleotide leader RNA sequence. This fragment was used directly for nick translation without subcloning. Nick translations of cDNA clones (0.5-l .O pg) were performed in 50 mlLl Tris-HCI (pH 7.8) 5 mM MgC12, 5 mlLl DlT, 50 pg/ml bovine serum albumin (BSA), 10 mM each of dATP, dGTP, and l'TP, 100 j&i [a-32P]dCTP (3200 Ci/mmol), 5Ob glycerol, 1 fig/ml DNase I (Worthington), and 2 U of DNA polymerase I (Boehringer Mannheim Biochem) for 2 hr at 15". Unincorporated nucleotides were removed by several precipitations with 80% ethanol. Hybridizations were performed at 42" for 36 hr in 50% formamide, 10 mM Na phosphate (pH 6.5) 5x SSC (1 X SSC = 0.15 A# NaCI, 0.015 1\/1 Na citrate, pH 7.0) 1 OX Denhardt's solution (1 X Denhardt's solution: 0.02% each of Ficoll, polyvinylpyrrolidone, and BSA) and 125 pg/ml salmon sperm DNA (Baric et a/., 1985) . Approximately 40-60 X lo6 cpm/pg of DNA were hybridized to each blot. After hybridization, the filters were washed, dried, and exposed to XAR films at -80" in the presence of an intensifying screen.

Equivalent amounts of intracellular RNA (25 or 50 rg) were separated by electrophoresis on 8% polyacrylamide gels in 0.1 M Tris-borate buffer (pH 8.4) containing 1 mM EDTA and 6 M urea. For analysis of small RNAs ranging between 30 and 150 nucleotides in length, the samples were electrophoresed at 1000 V (25-30 mA) until the bromphenol blue dye marker had migrated approximately 20 cm from the origin. To analyze larger RNAs ranging between 100 and 300 nucleotides in length, electrophoresis was at 1000 V until the xylene cyanol dye marker had migrated 35-40 cm from the origin. Following electrophoresis, urea was removed by twice washing the gel in ice-cold TAE buffer (0.04 M Tris-HCI, pH 7.4, 0.02 M Na acetate, and 0.001 M EDTA) for 15 min each, and the RNA was electroblotted to Zeta-probe paper (Bio-Rad) in cold TAE. Electroblotting was performed at 4" in a circulating chamber at 390 mA for 20-24 hr and an additional 2 hr at 1 A to ensure the transfer of larger RNAs above 500 nucleotides in length (Baric et a/., 1985; Stellwag and Dahlberg, 1980) . Following electrotransfer, the paper was gently washed in TAE and baked at 80" for 2 hr. Prehybridization was performed for 36 hr at 42" in 50% formamide, 10 mM Na phosphate (pH 6.5) 5X SSC, 1 OX Denhardt's solution and 125 pg/ml salmon sperm DNA.

Precise sizes of the small RNAs were determined by electrophoresis of Ml 3 mp 7 sequencing T-ladders in lanes adjacent to intracellular RNA preparations. Since electrotransfer oftentimes decreases the resolution of individual bands, complete sets of Ml 3 sequencing A, T, C, G ladders were also electrophoresed with each set of RNA preparations, fixed, dried, and separately exposed to XAR-5 film. The Ml 3-T ladders electrotransferred with RNA preparations were aligned with the A, T, C, G ladders to determine the exact sizes of small RNAs.

Computing stability of MHV RNA secondary structure Predictions for regions of secondary structure in MHV RNA were made using the Zucker RNA folding program through Bionet (Zucker and Stiegler, 1981) . Stability of individual hairpin loops within the MHV sequence was calculated from the thermodynamics of adding a base pair to a double-stranded helix at 25" as reported by

Tinoco et a/. (1973) and modified by Salser (1977) . More specifically, the energies for stacking of internal GU pairs were computed as follows: GU next to GC, -1.3 kcal/mol; GU next to AU or GU, -0.3 kcal/mol. In addition, -1 .O kcaI/mol was subtracted from loop structures containing U nucleotides (Tinoco et al., 1973) .

To determine the structure and origin of the small RNAs present in MHV-infected cells, we constructed several cDNA probes specific for different regions of the MHV genome. We chose three genes for this study, namely, genes A, D, and F. Gene A probably encodes an RNA polymerase, while genes D and F encode a nonstructural protein p14 and the El protein, respectively. Since gene A represents the 5'-most region of the genome, the small RNAs originating from this gene represent products of RNA replication. The other two genes represent two internal genes which have been well characterized (Armstrong eta/,, 1984; Skinner and Siddell, 1985) . The map positions of the probes used in this study are depicted in Fig. 1 .

The probe specific for leader region was derived from a gene F-specific clone (pHN42) which contains the entire leader sequence. The fragment (El-L) containing the first 84 nucleotides of MHV mRNA6 was used as the leader-specific probe in subsequent experiments (see Fig. 1 b) . The clones El -500 and El -300 were used to serve as the probes for the 5'-and 3'-ends of the gene F, respectively (Fig. 1 b) . The El -300 clone would not detect any leader-containing RNAs smaller than 534 nucleotides.

Clones specific for genes A and D were derived from cDNA clones as described under Materials and Methods.

To determine the structure and origin of the small RNAs present in MHV-infected cells, we first used a leader-specific cDNA probe to identify small RNAs terminated within or around the leader RNA sequence. These small RNAs should represent RNA species derived from the 5'-end of genomic or subgenomic mRNAs. These RNA species have been detected previously (Baric et a/., 1985) but their size and structure have not been precisely determined. Intracellular RNA was extracted from MHV-infected cells and separated by electrophoresis on polyacrylamide gels. Following transfer to Zeta-probe paper, the blot was probed with a leader-specific cDNA probe (El-L) (Fig. 1 b) . As shown in Fig. 2 , several distinct small RNAs ranging from 47 to 84 nucleotides were detected, similar to our previous a cBB\~~

D63pT P

FIG. 1. The structure of the MHV cDNA probes. Clone F82 contains the complete 5'-end of the genome and overlaps with clone C96. These clones were used to detect small RNAs originating during RNA replication (a). Clones D63X, El -500, and El -300 were used to detect small RNAs originating during transcription of mRNA 4 (gene D) and mRNA 6 (gene F) (b). Probe E 1 -L was used to detect small leader RNAs containing the leader sequence.

The exact sizes of these clones are described under Materials and Methods. Abbreviations used for cDNA fragments: B, BamHI; D, Ddel; E, EcoRI; H, Hindlll; P. Pstl; S, Seal; X, Xbal.

report (Baric eT a/., 1985) . The sizes of these RNA spe-Larger RNA species were also reproducibly detected cies were precisely determined from the accompanying by the leader-specific probe, consistent with previously T-ladder of Ml 3 sequences. Their sizes were repro-published results (Baric et a/., 1985); however, under ducible, as evident from the RNA patterns of two prep-the conditions used, they were not well resolved. These arations derived from different cell lines, although the leader-containing RNAs range from 110 to more than relative amounts of the various RNA species varied.

1000 nucleotides in length. To determine the structure The four smallest RNA species of 47, 50, 57, and 65 of these larger leader-containing RNA species, the RNA nucleotides correspond in size to the lengths between blots were hybridized with cDNA clone F82, reprethe 5'-end of the leader RNA and a region of two weak senting 3.8 kb of the sequences at the 5'-end of the overlapping hairpin loops around nucleotides 42-56 genomic RNA (Shieh et al., 1987) (Fig. 1 a) . Several disand 52-72 ( Fig. 3; Table 1 ) (Shieh et a/., 1987) . Thus, tinct RNA species ranging from 136 to larger than 281 these RNA species are likely to represent RNA tran-nucleotides were resolved (Fig. 4A) . It is notable that scriptional products terminating within the leader se-the two independently isolated preparations of intraquences. The larger leader-containing RNAs of 74 and cellular RNA gave identical RNA bands. When the clone 77 nucleotides, and occasionally an RNA of 84 nu-C96, representing the internal region of gene A (apcleotides, also correspond to the region of a hairpin proximately 3.8 kb from the 5'-end) (Fig. 1 a) , was used, loop at nucleotides 80-l 43 from the 5'-end of the ge-no distinct small RNA bands were detected (Fig. 4B ). nome and a postulated transcriptional termination sig-

The hybridization with C96 was generally weaker than nal (UUUAUAAA) for the MHV leader RNA synthesis that with F82, since the C96 probe would not detect (Shieh et a/., 1987) (Fig. 3) ""G%H c u g'-2 (Fig. 2) . The free energy of these hairpin loops is shown in Table 1 . data suggest that these RNAs represent the transcriptional intermediates derived from the 5'-end of the genomic RNA, and that transcription from the 5'-end of the genome terminates or pauses at distinct sites, The length of these RNAs correspond to the lengths between the 5'-end and the sites of potential hairpins (Table 1; see Discussion).

The leader-containing RNAs larger than 1 10 nucleotides in length detected by the leader-specific cDNA probe were more heterogeneous (Fig. 2) (Baric et al., 1985) suggesting that multiple RNA species in this size range were present. However, the probe representing the 5'-end sequences of gene A detected discrete species (Fig. 4A) , suggesting that the larger leader-containing RNAs may also represent RNA species derived from other regions of the genome during subgenomic RNA synthesis. Therefore, cDNA probes specific for the 5'-coding sequences of genes D and F were tested. The probe representing the 5'-end of the gene F (El-500) detected RNA species of 118, 123, 127, 143/l 45, 150, 158, 164, 169 , and two groups of poorly resolved RNAs ranging between 241-260 and 270-281 nucleotides in length (Fig. 5A ). These RNA species were different from those detected with the gene A probe (Fig. 4A ). In addition, they were not detected with a probe (El-300) representing the 3'-end of the gene between nucleotides 534 and 780 (Fig.  5C ). The gene D-specific cDNA probe (D63X) (Fig. 1 a) also detected a different set of small RNA species of 117, 135, 160, 168, 205, and 218 nucleotides in length (Fig. 58) . Smaller amounts of RNA of 173 and 199 nucleotides in length were also detected. These data suggest that transcription of subgenomic mRNAs also terminates or pauses at many sites. Computer models of RNA folding suggest the presence of hairpin loops in regions of nucleotides 105-122, 114-141, 123-167, 193-226 , and 242-285 within the sequence of mRNA6 (gene F) (Fig. 6b) and in the regions of nucleotides 106-l 31, 105-l 62, and 168-204 and 21 l-235 within mRNA4 (gene D) (Fig.  6a) . The predicted stability of these hairpin structures is shown in Table 1 . These data show that most of the small RNAs detected in MHV-infected cells have termination sites within the hairpin loop structures, suggesting a possible correlation between the generation of small RNAs and the presence of secondary structure in the template or product RNAs.

If the small RNA species detected in MHV-infected cells represent specific products released from the template RNA during transcriptional pausing, rather than degradation products of the mRNAs, then the small RNA species would be expected to be present in the RI structure which is involved in RNA transcription. Conversely, if RNA transcription proceeds in a continuous manner, the RNA species on the RI structure should be heterogeneous, without predominant RNA species. To distinguish between these two possibilities, 18-40 S RI RNA (Baric et a/., 1983; Sawicki and Sawicki, 1986) was isolated and analyzed for the presence of small leader-containing RNA using two probes, F82 and El -500, representing genes A and F, respectively. As shown in Fig. 7 , the small RNA species present on the RI RNA are the same as those present in the whole cell lysates. Furthermore, the 4 S RNA fractions also contain an identical set of small RNAs, indicating that some of the small RNAs were dissociated from the RI RNA. These results suggest that the small RNA species are true transcriptional intermediates.

Previous findings in our laboratory demonstrate that RNA recombination could occur at a very high frequency during a mixed infection with two strains of MHV (Makino et a/., 1986a). This high frequency is reminiscent of RNA reassortment described for viruses with segmented RNAs (Fields, 1981) and suggests that MHV replication might involve the generation of free and discontinuous RNA intermediates. These intermediates could participate in RNA recombination by a copy-choice mechanism. The detection of multiple discrete species of leader-containing RNAs, which range from 47 to more than 1000 nucleotides in length, in MHV-infected cells further supports this model (Baric et al,, 1985) . In this report, we examined the origin, structure, and mechanism of synthesis of these RNAs. The data suggest that these RNA species are likely to represent transcriptional pausing products of MHV RNA synthesis. The pausing sites correspond, in general, to the potential hairpin loops present in the template or product RNAs. This transcriptional pausing mech-anism is reminiscent of the transcriptional pausing of Q/3 phage RNA, in which RNA polymerase pauses in regions of hairpin loop structures (Mills et a/., 1978) . Pausing also occurs during DNA-directed DNA synthesis (Huang and Hearst, 1981; Sherman and Gefter, 1976) , DNA-directed RNA synthesis (Maizels, 1973; Rosenberg et al., 1978) and in reverse transcriptase reactions (Efstratiadis et al., 1975; Haseltine et al., 1976) . Similar to the mechanism of Qj3 RNA transcrip- tion, the majority of MHV small RNAs terminate at the 3'-side of hairpin loop structures in the product or template strands, which have free energies ranging from -1.6 to -77.1 kcal/mol ( Fig. 6 and Table 1 ). The stabilities of these hairpin loops are comparable to those of Q/3 (-9.8 to -23.8 kcal) and tRNA (-4.5 to -15.0 kcal) hairpin loops, whose existence in the RNA has been demonstrated by various physical and biochem-ical methods (Auron et al., 1982; Gehrke et al., 1983; Kramer and Mills, 1981) . Whether the pausing RNA intermediates of Qp or other pausing transcriptional model systems are dissociated from the RNA template is not known; however, the data presented in this paper clearly show that at least some of the MHV RNA intermediates are separated from the template RNA strand. It has previously been shown that the nascent RNA chains of G&I and MS2 phages are probably bound to the template strand only by short duplex regions (Weissman, 1974) . It is also possible that the MHV RNA polymerase is a nonprocessive enzyme, thus releasing some of the RNA intermediates after pausing. Currently, there is no direct evidence to prove that these free small RNAs actually rebind to the RNA template and participate in continuing transcription; however, the high frequency of RNA recombination (Makino et a/., 1986a) suggests that such rejoining does occur.

The recent isolation of several MHV recombinants with multiple crossovers further strengthened this probability (Keck et a/., 1987) . Most interestingly, the crossover sites in the two recently isolated recombinants, A-l and A-5, have been mapped within a region of the leader sequences (nucleotides 35-60 from the 5'-end) (Keck eta/., 1987) , where three small RNA species were detected (Fig. 2) . The isolation of these recombinants strongly suggests the functional roles of the pausing RNA intermediates. It is noteworthy that, during the transduction of the proto-oncogene c-fps by a retrovirus, right-handed recombination occurred near or in stable hairpin loop structures (Huang et al., 1986) . These data, coupled with the fact that reverse transcriptase "pauses" in regions of secondary structure (Haseltine et al., 1976) suggest a common mechanism of recombination for retroviruses and coronaviruses, involving paused transcriptional intermediates. In addition, it has previously been shown that short oligonucleotides can act as primers for RNA synthesis in vitro (Minkley and Pribnow, 1973; Niyogi and Stevens, 1965) . Thus, it seems likely that the free RNAs in MHVinfected cells could also be used for chain elongation. Recently, our laboratory has shown that the MHV leader RNA can act in Vans to participate in RNA transcription (Makino et al., 1986b) .

The four leader-containing RNA species of 65-84 nucleotides in length are the most likely candidates for the primers of subgenomic mRNA transcription, since they fall within the size range of the leader sequences present in the MHV subgenomic mRNAs (Lai et a/., 1984; Spaan et a/., 1983) . It is not clear, however, whether multiple leader RNAs are involved in priming mRNA transcription. According to our present model of leader-primed transcription, these leader RNAs would have to be cleaved before serving as primer (Shieh et a/., 1987) . These RNAs are likely generated due to the hairpin loop structures in nucleotides 52-72 and 80-l 43, and the intervening AU-rich sequences at the 5'-end of the genome (Fig. 3) (Shieh eta/., 1987) .

It should be noted that the pausing or termination sites of these small RNAs were determined by assuming that they were generated from correct transcriptional initiation sites. Although the extremely small amount of these RNAs makes it impossible to resolve this issue definitively, several reasons argue for the interpretation that they were initiated from the same site: first, the leader-primed transcription mechanism of MHV ( Baric et al., 1983; Shieh et a/., 1987) indicates that the same primer is used for the transcription of various mRNAs; second, these RNA species are very reproducible in different RNA preparations and are present on replicative intermediate RNA; third, RNA recombinants have been isolated whose recombination sites may be in an area corresponding to the termination sites of these small RNAs (Keck et a/., 1986) suggesting that these RNAs have biological functions; and finally, correct initiation was noted in a similar study with QB phages (Mills et a/., 1978) .

The biochemical and biological data presented here and elsewhere (Baric eta/., 1985; Makino eta/., 1986a) are, thus, consistent with a mechanism of MHV transcription which is discontinuous and nonprocessive (Fig. 8 ). In this model, the viral polymerase pauses in

Molecular Cloning-A Laboratory Manual

Optimal computer folding of large RNA sequence using thermodynamics and auxillaty information. Nucleic Acids Res. 9, 133-l 48.