0022-538X/78/0025-0274$02.00/0 JOURNAL OF VIROLOGY, Jan. 1978, p. 274-284 Copyright X 1978 American Society for Microbiology

Vol. 25, No. 1 Printed in U.S.A.

Endoribonuclease Activity Associated with Animal RNA Viruses DANIEL KOLAKOFSKY' AND SIDNEY ALTMAN2 Department of Microbiology, University of Utah Medical Center, Salt Lake City, Utah 84132,1 and Department of Biology, Yale University, New Haven, Connecticut 065202 Received for publication 27 June 1977

A specific endoribonucleolytic activity was detected when detergent-lysed vesicular stomatitis or Sendai virus was incubated with the precursor to Escherichia coli tRNAT-l. The cleavage products produced and the characteristics of the reaction were similar to those previously reported for human KB cell RNase NU. Like RNase NU, the virus-associated reaction generates 5'-hydroxyl and 3'phosphate groups at the cleavage sites. At protein concentrations similar to those used to test vesicular stomatitis and Sendai viruses, virions of Sindbis virus and poliovirus also exhibited endoribonucleolytic activity, but reovirus, simian virus 40, and minute virus of mice did not. This endoribonuclease may be of physiological relevance to some of the viruses we tested.

Sendai virus (SV) and vesicular stomatitis virus (VSV) are single-stranded RNA viruses (15, 46). They are sometimes called "minusstrand" viruses because their genomes are transcribed into complementary RNA sequences, which serve as mRNA (6). Several virus-specific monocistronic mRNA's, capped at their 5' termini, can be isolated from infected cells (28, 29, 35, 37). It is not known whether these mRNA's are transcribed as monocistronic segments or are cleaved by endoribonucleolytic action from a longer "plus"-strand transcript. If 5'-phosphate groups are a prerequisite for the mRNA capping reaction (21) and if the mRNA's result from the cleavage of a longer plus-strand transcript, then an endoribonuclease that generated 5'-phosphate groups could be responsible for the presumed cleavage step (1). Such an enzymatic activity might be similar to RNase P, a tRNA precursor processing enzyme, which has been identified in extracts of Escherichia coli (5, 39), human tissue culture KB cells (30), tissue cultured monkey kidney cells (4), and chicken embryonic tissue (E. J. Bowman and S. Altman, unpublished data). RNase P generates 5'-phosphate termini in its cleavage products. We thus embarked on a search for this enzyme in SV and VSV virions, using the well-characterized precursor RNA to E. coli tRNAT`r as a probe substrate (2, 5), We did not find evidence of RNase P activity in these virions, but, to our surprise, they do contain an activity very similar to RNase NU, which has been identified in extracts of KB and monkey and kidney cells (9; A. L. M. Bothwell, Ph.D. thesis, Yale University, New Haven, Conn., 1975). RNase NU produces

fragments containing 5'-hydroxyl and 3'-phosphate groups in several RNA molecules which are thought to turn over rapidly in vivo (9, 10), one of which is the precursor to E. coli tRNATyr. In this report we describe the properties of the virus-associated endoribonuclease and show its similarity to KB cell RNase NU. We also show that other single-stranded animal viruses have associated endoribonucleolytic activity but that two DNA viruses and a double-stranded RNA virus have no activity.

MATERIALS AND METHODS Virus and nucleocapsid preparation. SV was grown on LLCMK2 (rhesus monkey kidney) cells. The virus was recovered from the tissue culture medium by polyethylene glycol precipitation and centrifuged to equilibrium on 10 to 50% (wt/wt) sucrose gradients containing 10 mM Tris-hydrochloride (pH 7.4), 1 mM EDTA, and 0.5 M NaCl (TNE). The virus band was then diluted fivefold with TNE, pelleted in the ultracentrifuge, and resuspended in 10 mM Tris-hydrochloride (pH 7.4) and 1 mM EDTA. VSV was grown in either chicken embryo fibroblasts or HeLa cells. T'he chicken embryo fibroblast-grown virus (a gift of B. Brown, University of Utah) was purified by using the method described above for SV. The HeLa-grown virus (a gift of S. Humphries, University of Utah) was banded in 10 to 50% potassium tartrate gradients in addition to being subjected to the purification scheme outlined above. Sindbis virus (a gift of J. Etchison, University of Utah) was grown in BHK-21 cells and purified according to Sefton and Keegstra (43). Poliovirus (a gift of E. Ehrenfeld, University of Utah) was grown in HeLa cells and purified according to Celina and Ehrenfeld (14). Nucleocapsids of each virus were prepared as described by Breindl and Holland (12), with the modifications noted in the appropriate figure

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legends. Reovirus (a gift of L. Ratner, Yale University) was purified as described by Graziadei and Lengyel (26). Simian virus 40 (SV40) (a gift of W. Beattie, Yale University) was purified as described by Estes et al. (20) and Lake et al. (31). Minute virus of mice (a gift of D. Ward, Yale University) was purified according to Tattersall et al. (44). RNase preparation. KB cell RNase NU, prepared as described by Bothwell and Altman (9), was used in the experiments described in this report. The RNase NU (a gift of R. Koski, Yale University) was derived either from a nuclear sap fraction (generated from the supernatant of a nuclear lysate) or from ribosome washes. Ribosomes from E. coli, washed according to the procedures of Schedl et al. (42) and containing both RNases P and P2 activity, were used in some experiments. An S-100 fraction from E. coli, containing RNase P activity (a gift of B. Stark, Yale University), was also used in one experiment. Radioactive polynucleotides. 3P-labeled radioactive RNAs were extracted from E. coli as described in the given references: precursor RNA to tRNATyr (129 or 96 nucleotides long) (5, 41; Bothwell, Ph.D. thesis; P. Schedl, Ph.D. thesis, Stanford University, Stanford, Calif., 1975); bacteriophage 080-induced M3 RNA (10, 36). Preparation of 5'-terminally labeled 32P-tRNA. Dephosphorylated bulk E. coli tRNA (1 ,ug), prepared according to Brownlee (13), was incubated with 6 U of authentic T4 polynucleotide kinase (Biogenic Research) and 10,uCi of [-y-3P]ATP (600 Ci/mmol; New England Nuclear) in a final volume of 100 pl, under conditions described by Bigger et al. (8). The reaction was stopped by the addition of 0.5 ml of 0.2 M sodium acetate (pH 5.5), 20 pl of 0.4 M EDTA-Na2, and 40 ytg of bulk E. coli tRNA. The final mixture was precipitated three times from 2.5 volumes of ethanol and then dialyzed extensively versus 1 mM Tris-hydrochloride, pH 7.8. The final specific radioactivity was approximately 30,000 cpm/Lg of tRNA.

Fingerprint analysis. Fingerprint analyses were

carried out by using the methods of Sanger et al. (40) as applied by Goodman et al. (24). Assays for RNase activity. Assays for RNase activity were carried out in a standard mixture (about 104 cpm of RNA substrate; protein containing enzymatic activity in the amounts shown in the figure legends; 30 mM Tris-hydrochloride [pH 8.0]-100 mM NH4Cl-5 mM MgCl2-10-4 M ,8-mercaptoethanol-10-4 M EDTA) as described by Robertson et al. (39) and Bothwell and Altman (9), except that the volume of the reaction mixture was 30 pi. Incubation was for 40 or 60 min at 370C. Reactions were stopped by adding 10 pl of the following solution to the reaction mixture: 40 mM EDTA and 0.01% bromophenol blue-50% sucrose-20% sodium dodecyl sulfate (100:80:20). The stopped reaction mixture was then layered directly on a 10% polyacrylamide gel for analysis unless otherwise indicated. When virus was used as the source of enzyme, it was sometimes lysed first in a 1% detergent solution (Nonidet P-40, Triton N-101, or Triton X-100), as described in the figure legends. Protein concentration determinations. The methods of Bradford (11) and Lowry et al. (33) were used for protein determinations.

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Analysis of RNase reactions of diethylaminoethyl ion-exchange paper. Samples from completed reaction mixtures (taken before addition of the sodium dodecyl sulfate stop mixture) were spotted on diethylaminoethyl paper (Reeve-Angel) and subjected to electrophoresis in pyridine-acetate buffer (pH 3.5) as described by Brownlee (13) for second digests of RNase Ti-generated fragments of RNA. This method allows separation of most tetranucleotides and smaller products from higher-molecular-weight material. The paper was autoradiographed after completion of electrophoresis, and the appropriate spots were cut out of the paper and assayed for radioactivity.

RESULTS Presence of an endoribonuclease in VSV and SV. VSV and SV, lysed with detergent, exhibit an endoribonucleolytic activity (Fig. 1) when assayed with tRNATyr precursor (129 nucleotides) as substrate. Untreated virus also exhibits this activity, but since the virus suspensions have been frozen and thawed many times, it is likely that they are already partly lysed. The SV cleavage is apparent only at the higher concentration used. We have found that VSV suspensions generally have a higher specific activity than SV suspensions. Nucleocapsid cores prepared from both viruses also have RNase activity (Fig. 1, lanes 6 and 7), but the VSV cores exhibit much more activity than the SV cores prepared from viruses grown in chicken embryo fibroblasts. The latter give a low but significant level of activity when compared with the control (Fig. 1, lane 8). SV cores derived from viruses grown in monkey kidney cells have much higher RNase activity (see below). Cleavage by the VSV RNase activity increases with time of incubation at 370C for at least 40 min (Fig. 2). It is also sensitive to proteolytic inactivation and is proportional to the amount of virus added (data not shown). The effects of various ions on the reaction (Fig. 2) are discussed further below. In parallel experiments with authentic KB cell RNase NU or E. coli RNases P and P2 (42) (see Fig. 3 and 4) cleavage patterns identical to those produced by the virus-associated RNase are seen only when RNase NU is used. The cleavage products generated by the SV-associated RNase with the 080-coded M3 RNA (36) are also similar to those made by RNase NU (11), as judged by their relative mobilities (Fig. 4). The equimolar production of large and small fragments generated from these substrates is good evidence that the association of the RNase activity with the viruses is highly specific, since extensive cell fractionation is required before KB cell RNase NU is sufficiently purified, so that the smaller cleavage fragments are recovered quantitatively. However, the major

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FIG. 1. Virus-associated endoribonuclease activity. Samples of virus (after treatment with 1% Nonidet P40) or nucleocapsid suspensions were incubated in the standard reaction mixture containing about 104 cpm of tRNAyr precursor (129 nucleotides) as substrate, mixed with the stopping solution, and layered on an 8% polyacrylamide gel as described in the text. Lane 1, Sendai virus grown on monkey kidney cells, 8.5 pg; lane 2, as in lane 1, 17 ug; lane 3, blank; lane 4, VSV grown on HeLa cells, 30.5 pg; lane 5, VSV grown in chicken embryo fibroblasts, 52 jig; lane 6, Sendai nucleocapsids prepared from virus grown in chicken embryos as described in the text, c30 ,ug; lane 7, VSV nucleocapsids prepared from VSV grown in HeLa cells as described in the text, '35 ig; lane 8, no enzyme added; lane 9, marker 4S RNA extracted from Chinese hamster ovary cells. In none of the experiments described in this report was the final concentration of detergent in the reaction mixture more than 0.3%.

cleavage product generated with the shorter (96 nucleotides [41]) tRNAT" precursor (Fig. 4, lane 9) has a somewhat faster mobility than the RNase NU-generated product (Fig. 4, lane 11) and is similar to the product generated by VSV when it is present at high concentrations (Fig. 1, lane 5). In this reaction, which is discussed further below, no additional large oligonucleotide, which might be generated as a result of a single endonucleolytic cut, is detected in the gel. To test whether or not the virus-associated RNase activity was an integral part of the macromolecular structure or loosely bound to the outside of the virions, we fractionated VSV virions after disruption with Nonidet P-40, using the method of Breindl and Holland (12). Three fractions of disrupted viral material were taken from the step gradient: a pellet fraction, and the

visible material present at both the top and middle interphases of the gradient. Sodium dodecyl sulfate-polyacrylamide gel analysis (Fig. 5, right) showed that both the pellet and middle fractions consisted almost entirely of the L, NS, and N proteins (Fig. 5, right panel, lanes B, C), those proteins associated with the viral nucleocapsids, whereas the top fraction consisted almost entirely of the G and M proteins (Fig. 5, right panel, lane D), those proteins associated with the viral envelope. Each fraction was also tested for RNase activity. Figure 5 (left panel) shows that only the pellet fraction contained RNase activity. The VSV used in this experiment had already undergone two cycles of equilibrium centrifugation designed to remove adhering cellular contaminants during its purification. The experiment illustrated in Fig. 5 dem-

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onstrates that the RNase activity associated with the virus is an integral part of the virus structure and not associated with membranebound contaminants, since these do not cosediment with the pellet fraction. However, since all the RNase activity was associated with the pellet fraction (which does contain virion transcriptase activity [12] and less than 1% of intact virus from the layered sample) whereas most of the viral nucleocapsids were found in the middle fraction, RNase activity appears to be associated with a fast-sedimenting subpopulation of nucleocapsids. Nucleocapsids prepared from SV grown on 1 2

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monkey kidney cells also show RNase activity and the absence ofmembrane-associated protein (data not, shown). These nucleocapsids have much more RNase activity than those prepared from SV grown in chicken embryos (Fig. 1, lane 6) and also have much more transcriptase activity (D. Kolakofsky, unpublished data). SV virions from the two sources also show correspondingly different levels of RNase activity. It could be that the exact nature of all the molecules packaged by the virions may vary in different tissue sources and thus differentially affect the amount of RNase activity in the intact virions. Virus-associated RNase is similar to 7

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FIG. 2. Kinetics and some ion requirements of the VSV-associated activity: reactions were carried out as described in the legend to Fig. 1 and in the text. Analysis was on a 10%l polyacrylamide gel. Lane 1, E. coli RNase P, 0.5 jig; lanes 2-4, a reaction mixture, scaled up threefold in volume with 65 ,ig of total VSV, was prepared and samples were taken at various times after incubation: 0 min (lane 2), 20 min (lane 3), and 40 min (lane 4); lanes 5-7, the same as lanes 2-4 except 195 jLg of VSV was in the reaction mixture (at this higher VSV concentration, the initial kinetics of the reaction are not appreciably changed); lane 8, VSV, 15 pg; lane 9, VSV, 15 jg, no NH4CI or MgCl2 in the reaction mixture; lane 10, VSV, 15 jig, no NH4Cl in the reaction mixture; lane 11, VSV, 15 jig, no MgCl2 in the reaction mixture; lane 12, same as lane 1; lane 13: marker KB cell RNA.

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gel slices and the RNase T1 digestion products were subjected to fingerprint analysis. The resulting fingerprints (Fig. 6) were identical to those published previously for the cleavage products generated by KB cell RNase NU (9). A significant result of this experiment is that the virus-associated RNase generates, as does RNase NU, 5'-hydroxyl and 3'-phosphate groups at its cleavage sites. The VSV-associated RNase was extensively tested and showed the same ionic requirements as RNase NU (Table 1), with the exceptions

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FIG. 3. Comparison of VSV-associated RNase and KB cell RNase NU: reactions were carried out with Nonidet P-40-treated virus and KB cell RNase NU (see text and the legend to Fig. 1) and layered on 10% polyacrylamide gels for analysis. Lane 1, No enzyme; lane 2, VSV, 45 pg; lane 3, VSV, 15 jig; lane 4, VSV, 45 pig, no magnesium ion in mixture; lane 5, KB RNase NU, 0.35 ug; lane 6, KB RNase NU, 0.35 jg, no magnesium ion in mixture; lane 7, E. coli ribosomes washed in 0.5MNH4CI containingRNases P and P2 activity, 30 pg.

RNase NU. Other criteria in addition to mobility of cleavage products can be used to determine if the virus-associated RNase is identical to RNase NU. These include fingerprint analysis of the cleavage products and ionic requirements of the reaction. To prepare sufficient quantities of the cleavage products labeled bands 1, 2, 3, and 4 in Fig. 3 for fingerprint analysis, reactions with VSV were scaled up ninefold and the products were separated in a polyacrylamide gel. The cleavage products were extracted from the appropriate

FIG. 4. SV-associated RNase characteristics with various substrates: reactions were carried out as described in the text and analyzed in 10% polyacrylamide gels. The SV was not treated with detergent before use. Reactions analyzed in lanes 1-6 contained tRNA7r precursor (129 nucleotides) as substrate. Lane 1, No enzyme; lane 2, SV, 3.4 ug; lane 3, SV, 10.2 pg; lane 4, SV, 17 pg; lane 5: SV, 3.4 pg, 0.2 M NH4Cl in reaction mixture; lane 6, SV, 3.4 pg, no NH4Cl in reaction mixture; lane 7, SV, 10.2 pg, M3 RNA substrate; lane 8, no enzyme, M3 RNA substrate (there is some overflow from lanes 7 and 8 into adjacent lanes); lane 9, SV, 10.2 pg, tRNATYr precursor (96 nucleotides) substrate; lane 10, no enzyme, tRNATr precursor (96 nucleotides) substrate; lane 11, KB cell RNase NU, 0.28 pg, tRNATyr precursor (96 nucleotides) substrate.

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FIG. 5. RNAase activity associated with VSV nucleocapsid preparations: preparations were assayed as described in the text and the legend to Fig. 1 and layered on a 10% polyacrylamide gel for analysis. Nucleocapsids were prepared as follows: 190 pg of VSV (HeLa) was disrupted in 0.1 ml of 0.5% Nonidet P-40 (NP-40), 50 mM Tris-hydrochloride (pH 8.0), 5 mM MgC42, 0.1% /3-mercaptoethanol, and 0.25 M NaCl at 0°C according to Breindl and Holland (12). The disrupted virus was layered onto a step gradient of 0.25 ml of 75% (vol/vol) and 0.25 ml of 30% glycerol in 10 mM Tris-hydrochloride (pH 8.0) and centrifuged for 1 h at 40,000 rpm (2QC) in the Spinco SW50.1 rotor with adaptors to hold 0.6-ml centrifuge tubes. After centrifugation, the visible band at the interphase between the 30% glycerol and the top of the gradient (top band) was collected in 50 ,il, and the visible band between the 30 and 75% glycerol solutions (middle band) was collected in 75 p2. The remaining solution was removed, the centrifuge tube was allowed to drain upside-down for 5 min, and the bottom pellet was resuspended in 25 Al of 10 mM Tris-hydrochloride, pH 8.0. (Left panel) Lane 1, VSV nucleocapsid pellet suspension, 5 p2; lane 2, "middle band," 15 p2; lane 3, "top" band, 10 /pl; lane 4, VSV made 1% in NP-40 before use, 7.5 pg; lane 5, as in lane 4 but no NP-40 added; lane 6, no enzyme; lane 7, Sindbis virus (made 1% in NP-40 before use), 7pg. (Rightpanel) sodium dodecyl sulfate-10%polyacrylamide gel analysis of various fractions stained with Coomassie brilliant blue: lane A, 20 pg of undisrupted VSV; lane B, VSV nucleocapsid pellet suspension, 7 id; lane C, "middle" band, 20 p1; lane D, "top" band, 12p2.

noted below. The SV-associated RNase was tested only for the ammonium and magnesium ion requirement and showed the same response in this regard as the VSV-associated RNase. With respect to the VSV reaction, it was inhibited 30% by 1 ,ug of Penicillium chrysogenum double-stranded RNA in the reaction mixture and about 70% by 16 ,tg of E. coli bulk tRNA in the mixture. There was little detectable activity when phosphate was the anion in the mixture. Compared to pH 8.0, the extent of the reaction at pH 7.5 was 45%, and at pH 7.0 it was 20%. Unlike the result previously reported for RNase NU (9), both virus-associated RNases showed a requirement for magnesium ion. In fact, the reaction seemed optimal when 8 to 10 mM MgCl2 was present in the reaction mixture. The enzyme, when associated with the virus, may be affected in such a way by the other viral

proteins that a divalent cation may become necessary for the RNase NU-like reaction to proceed when the enzyme is in the viral complex. Another subtle difference in the virus-associated reaction compared with the reaction of RNase NU prepared directly from KB cells appears to be the kinetics of cleavage of bacteriophage 080 M3 RNA (10). Whereas cleavage products with identical mobilities appear to be produced by each enzymatic activity, the kinetics of cleavage at each site, and thus the relative proportion of the cleavage products from each reaction, does not appear to be the same when the two reactions are analyzed in the same gel (R. Koski and S. Altman, unpublished data). Reaction products in the presence of excess virus. In the gel analysis of preparative cleavage reactions with intact (129 nucleotides) tRNA"yr precursor, a minor cleavage product

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FIG. 6. Fingerprint analysis of cleavage products of tRNAT-Vr precursor (129 nucleotides) generated by VSV-associated RNase activity. Cleavage products (see Fig. 3), after extraction from polyacrylamide gels, were digested with RNase Tb, and the products were separated in two dimensions as described by Brownlee (13). First dimension, Pyridine-acetate (pH 3.5), right to left; second dimension, 70/o formic acid, top to bottom. (A) Uncleaved tRNATYr precursor (129 nucleotides); (B) band 1; (C) band 4; (D) cleavage product produced at high virus concentration (see text) (the arrow points to the area of the fingerprint where pGp, absent here, is seen in analyses of mature tRNATvr); (E) band 2; (F) band 3.

containing about 10% of the total cleaved material was apparent in the region close to, but moving slightly faster than, band 2. This product becomes especially evident when an excess of virus-associated RNase is present in the reaction mixture, as can be seen in Fig. 1 (lane 5), Fig. 3 (lane 3), Fig. 4 (lane 3), Fig. 5 (lane 7), and Fig. 7 (lanes 3 and 4). Fingerprints of this cleavage product of the 129-nucleotide precursor, and of the major product of cleavage of the 96-nucleotide precursor (Fig. 4, lane 9), are identical to each other and to that of a cleavage product containing the mature tRNATy, sequence, except that the 5'-phosphate terminus is absent (39). In our experiments, the 5' terminus is OHG and extra 3'-terminal nucleotides are present. This product may be a consequence of secondary

reactions of the virus-associated RNase. Such reactions, which may become apparent only with certain substrates and/or under certain conditions, are also manifested by E. coli RNase III (18). Alternatively, the new cleavage product could be generated by RNase NU action followed by a 5'- to 3'-exonuclease activity, producing 5'-hydroxyl and 3'-phosphate groups (22), which stops at the beginning of the tRNA secondary structure. However, when the products of the reaction wth excess virus are subjected to electrophoresis on diethylaminoethyl ion-exchange paper (see Materials and Methods), we recover di-, tri-, and tetranucleotides as well as mononucleotides. So it is possible that at high virus concentration band 2 is produced first and the additional degradation is carried out by fur-

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ther, or secondary, RNase NU endonucleolytic action rather than by novel exonucleolytic action. A third explanation for the formation of the new cleavage product is action by an RNase Plike activity accompanied by a 5'-phosphatase action. In this case, an additional 41-nucleotide fragment should be visible in our gel autoradiographs, but we never found it. On the other hand, using 5'-terminally labeled tRNA as substrate, we have detected a VSV-associated 5'monophosphatase activity (Table 2). Other viruses. Other viruses besides SV and VSV have associated RNase activity, which, as judged by electrophoretic mobilities of the cleavage products generated, appears similar to RNase NU. Sindbis virus and poliovirus, both of which have RNA genomes, exhibit RNase activity (Fig. 5 and 7). When some other viruses

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were used in our assay at the same protein concentration as that at which VSV, SV, Sindbis virus, or polioviruses might be expected to show nucleolytic activity with linear kinetics (300 ,ug/ml), detectable amounts of activity are observed with reovirus and SV40. A maximum estimate of the relative specific activity of RNase activity associated with reovirus or SV40 is 30-fold less than that found in VSV. If this calculation (made using data regarding the radioactivity found in residual intact substrate and cleavage products cut out from gel a.nalyses of in vitro reactions) were normalized on a viral particle basis rather than on a viral protein basis, the relative content of

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FIG. 7. RNase activity associated with Sindbis and polioviruses. Reactions were carried out as described in the text and the legend to Fig. 1 and analyzed in a 10% polyacrylamide gel. Lane 1, Marker 4S RNA extracted from Chinese hamster ovary cells; lane 2, Sindbis virus (made 1% in Triton N-101 before use), 7.5 Ag; lane 3, poliovirus (made 1% in Triton N-101 before use), 28 1ug (the rapidly migrating band near the bottom of the gel consists of mono- and small oligonucleotides resulting from the RNase reaction at high virus concentration [see text]); lane 4, same as lane 3 with no Triton treatment; lane 5, same as lane 4 but 5.6 aug of virus; lane 6, no enzyme.

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and minute virus of mice, have little or no activity. These observations taken together show that the association of the RNase with the virions is KB Activity RNase Additions Source of probably not a nonspecific aggregation phenom(% NU acRNase enon during intracellular virus growth. On the tivityb other hand, the physiological relevance of the 0 VSV or SV None RNase activity to, for example, minus-strand 54 + VSV NaCl, MgCl2 RNA virus replication is not immediately ap++ 100 VSV or SV NH4Cl, MgCl2 parent from the data presented here. Should ++ 70 VSV CaC12 8 ++ the RNase NU-like activity function to cleave VSV or SV NH4 Cl 27 VSV or SV MgCl2 the viral plus strand before capping, it is possible 10 VSV NaH2PO4, MgCl2 that at least one other enzymatic activity, a + 44 SV NH4Cl(0.2 M), MgCl2 polynucleotide kinase, must act on the cleavage a VSV or SV (about 15 pg/reaction mixture) was assayed products before capping, since RNase NU genas described in Materials and Methods. The basic mixture contained 0.03 M Tris-hydrochloride (pH 8.0), 10-4 M EDTA, erates 5'-hydroxyl termini. We have assayed for and 10-4 M fi-mercaptoethanol. The various substances listed such a virus-associated polynucleotide kinase, under "Additions" in the table were present in the following under the same conditions that allow authentic concentrations unless noted otherwise: 5 mM MgCl2; 100 mM phage T4 polynucleotide kinase function (38), NH4Cl; 100 mM NaCl; 100 mM NaH2PO4 (pH 8.0); 10 mM CaC12. Relative activity in our experiments was judged by and have not yet been able to detect it. Isolation of intact positive strands to use as cutting out gel bands of the major cleavage products and uncleaved substrate, assaying the radioactivity by Cerenkov test substrates for RNase NU is of obvious imradiation, and calculating the percent cleavage by using the portance, but this has so far proved elusive. It results of this assay. A rough equivalency for the extent of reaction with the data shown in the KB RNase NU column may be that a knowledge of the ionic requireis the following: -, 0 to 25%; +, 25 to 50%; ++, 50%. The ments of RNase NU will aid in this task. For extent of cleavage in the reaction mixture containing NH4Cl example, transcriptase reactions, which are norand MgCl2 was taken as 100%. mally carried out in vitro under conditions very bData taken from Bothwell (Ph.D. thesis) and Altman and similar to those of the RNase NU assay, could Bothwell (9). TABLE 1. Ionic requirements of the virus-associated RNase reactiona

RNase activity in reovirus, SV40, or minute TABLE 2. Phosphatase activity associated with virions and nucleocapsidsa virus of mice would be 100-fold or less than that found in VSV. This could represent an upper cpm re32Ptained Source of protein (amt added, /g) limit to contamination by cellular nucleases that have copurified with the viruses. No added protein ....... 12,116 E. coli alkaline phosphatase (1) 786 DISCUSSION 694 . E. coli alkaline phosphatase (4) .. .. 9,554 An endoribonuclease, with cleavage specificity SV (6) ..... 8,558 and ionic requirements similar to those of RNase SV (18) ... 10,872 (1) ................. NU, is found associated with SV and VSV and VSV ...... 9,450 (4) ..... cores. The association seems extremely specific VSV Sendai nucleocapsids (0.5) ... 9,836 in the sense that only RNase NU-like activity Sendai 8,490 nucleocapsids (2) .. is seen with the substrates we have used. All of No added protein (not incubated) 12,856 the viruses we have tested have been extensively assay was carried out in the same fashion as purified from cell extracts with procedures that thata The endoribonuclease activity. The substrate was usually involve two sedimentations in high-mo- bulk for tRNA terminally labeled with 3P, as described larity salt solutions as well as other treatments in Materials and Methods. Incubation was for 40 min designed to remove cell debris from the virus at 370C. In separate experiments with VSV we have particles (treatment with 1% sodium dodecyl shown that the reaction proceeds linearly for about sulfate in the case of poliovirus). Viruses that 40 min at 370C, that the rate increases with increasing are extruded from cells by budding may be ex- viral protein concentration, and that neither VSV nor pected to pick up host enzymes associated with SV virions exhibit any endonucleolytic or exonucleohost cell membranes (45). We have found the lytic cleavage of tRNA or 5S RNA. Retention of in high-molecular-weight material was RNase NU-like activity in budding (SV, VSV, radioactivity using the diethylaminoethyl paper ion-exand Sindbis) as well as nonbudding viruses (po- measured technique of Altman and Lerman (3). Values lio). In addition, the RNase activity can be iden- change of duplicate samples fall within 5% of their average. tified in nucleocapsid preparations from SV and This method was checked by electrophoreses of test VSV, although we cannot unequivocally say that reaction mixtures on diethylaminoethyl paper at pH the enzymatic activity resides in nucleocapsids 3.5 in pyridine-acetate buffer, as described in Materials per se. Three viruses we tested, reovirus, SV40, and Methods.

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be performed at lower pH or in the presence of phosphate ion in the hope of selectively inhibiting nuclease action. Some RNA species, like tRNA's (17, 19, 27), and other enzymes, like RNase H (7, 23, 25, 32, 34), are packaged by several viruses, but not all these companion molecules have an obvious role in any particular viral replication scheme (16). It is noteworthy that several single-stranded RNA viruses exhibit the endoribonuclease activity but a double-stranded RNA virus and two DNA-containing viruses have no significant level of this activity. The single-stranded viruses have one common feature: they all replicate through an intermediate containing a complementary stralid. It is possible that the associated endoribonuclease activity is involved in events at the 5' termini of the replicative intermediates rather than in mRNA processing, as discussed above. RNase NU can attack three procaryotic RNAs in single-stranded regions adjacent to regions that can be drawn as hairpin structures (9, 10). It was suggested that this may be a distinguishing feature of molecules, or parts of molecules, which are destined to turn over relatively rapidly in vivo. More recently, it has been shown that HeLa cell hn RNA is susceptible to attack by KB fractions that exhibit only RNAase NU activity, when assayed separately with tRNAT3r precursor as substrate (Koski and Altman, unpublished data). This enzyme must be capable of distinguishing not only RNAs that turn over rapidly and those that do not, but also among various classes of rapidly turning over mRNA's. It will be of certain interest to learn if RNase NU does perform an essential function in RNA virus replication as a processing or degradative enzyme. Of no less interest will be an understanding of how viruses package this enzyme with such selectivity. ACKNOWLEDGMENTS We thank A. M. Korner and R. A. Koski for help with some experiments. H. D Robertson and D. C. Ward kindly provided their comments on a draft of the manuscript. S. Altman wishes to thank F. Ruddle for use of his viral containment facility. Lila Kort provided excellent technical assistance.

This work was supported by grant PCM 75-2103 from the National Science Foundation to D.K. and by Public Health Service grants AI 12921 from the National Institute of Allergy and Infectious Diseases and GM 19422 from the National Institute of General Medical Sciences to D.K. and S.A., respectively.

ADDENDUM IN PROOF We have recently found that rat liver RNase inhibitor (A. A. M. Gribnau, J. G. G. Schoenmaker, and H. Bloemendal, Arch. Biochem. Biophys. 130:48-52, 1969) can completely inhibit VSV-associated RNase NU in vitro. C. M. Preston and J. F. Szilyagi (J. Virol. 21:1002-1009, 1977) have found

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