Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2037-2042, March 1996 Biochemistry

Characterization of small nontranslated polyadenylylated RNAs in virus-infected cells (inhibition of synthesis/in vitro /in vitro translation/in vivo polyadenylylation of tRNAs and small nuclear RNAs) CHUNXIA Lu AND ROSTOM BABLANIAN* Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 Communicated by Howard L. Bachrach, U.S. Department ofAgriculture, Greenport, NY, September 28, 1995 (received for review June 22, 1995)

ABSTRACT Host protein synthesis is selectively inhibited uninfected, infected-with-VV or infected-with-UV-irradiated in vaccinia virus-infected cells. This inhibition has been associ- VV (9600 ergs/mm2) HeLa spinner cells. Poly(A)+-containing ated with the production of a group of small, nontranslated, mRNA was isolated from total cytoplasmic RNA by oligo(dT)- polyadenylylated RNAs (POLADS) produced during the early cellulose chromatography. POLADS were isolated as de- part of virus . The inhibitory function of POLADS is scribed (10). associated with the poly(A) tail of these small RNAs. To deter- Construction of Plasmids That Contain POLADS. The mine the origin of the 5'-ends of POLADS, reverse transcription cDNA of POLADS was obtained by reverse transcription (13) was performed with POLADS isolated from W-infected cells at using a cDNA synthesis kit (United States Biochemical) and 1 hr and 3.5 hr post infection. The cDNAs ofthese POLADS were cloned into pBS or pBluescript KS 11 +/- at the Sma I site. cloned into plasmids (pBS or pBluescript H KS +/-), and their In Vitro Transcription. Constructs were linearized by Xba I nucleotide composition was determined by DNA sequencing. The or Kpn I, and in vitro transcription was done (14) in the results of this investigation show the following: There is no presence of T3 or T7 RNA polymerase, depending on the specific encoding for POLADS. The 5' ends ofPOLADS may orientation of the inserted cDNA of POLADS. be derived from either viral or cellular RNAs. Any RNA sequence Measurement of Protein Synthesis in Vitro. In vitro protein including tRNAs, small nuclear RNAs and 5' ends ofmRNAs can synthesis was done in reticulocyte lysate (15) treated with become POLADS if they acquire a poly(A) tail at their 3' ends micrococcal nuclease (16). Twenty-five-microliter reaction during infection. This nonspecific polyadenylylation found in mixtures were programmed with virus or HeLa mRNA, and vaccinia virus-infected cells is probably conducted by vaccinia were labeled with [35S]methionine (7). virus poly(A)+ polymerase. No consensus sequence is found on Determination of the Origin of 5' Ends of POLADS. Cloned the 5' ends of POLADS for polyadenylylation. The 5' ends of POLADS were sequenced with modified T7 polymerase Se- POLADS have no direct role in their inhibitory activity ofprotein quenase (17). The reaction was done on alkaline-denatured synthesis. double-stranded plasmid templates (18), and either a T7 primer or a reverse primer was used in the reactions. The DNA During vaccinia virus (VV) infection, a population of small sequences of the POLADS were compared with the nucleotide nontranslated polyadenylylated RNAs (POLADS) are pro- sequence database provided by the National Center for Bio- duced. The relation between the production of POLADS and technology Information using the BLAST network service. the selective inhibition of cell protein synthesis has been exten- Construction of Artificial POLADS. Poly(C) and rRNAs sively studied (1-10). POLADS selectively inhibit host protein were hydrolyzed with 0.2 M NaOH for 10 min at 37°C and synthesis (3-7), and the inhibitory moiety has been shown to neutralized with HCl (0.2 M). These RNAs were precipitated reside only on their poly(A) tails (5, 6, 10). The longer the poly(A) with 100% ethanol and electrophoresed on a thin (0.4 mm) tails, the greater the inhibitory activity of POLADS (5, 6, 10). 5% acid/urea/polyacrylamide gel. The 80- to 150-nt-long To further characterize these inhibitors, we cloned POLADS RNAs were eluted from the gel in 0.5 M NH4 (OAC), 10 mM and determined their composition. The results in this investiga- EDTA, and 0.1% SDS at 37°C for 18 hr. Polyadenylylation tion demonstrate that during VV infection, both viral or host of these RNAs (rRNA, poly(C), and tRNA) was done in the mRNAs and, in addition, host tRNA and small nuclear RNA are presence of Escherichia coli poly(A) polymerase. In 100-,l polyadenylylated and may serve as POLADS. Polyadenylylation reaction mixture, there were 8 units of poly(A)+ polymerase, of these normally nonpolyadenylated host cell RNAs is probably 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM MgCl2, 5 conducted by VV poly(A) polymerase that is released from the mM MnCl2, 2 mM ATP, bovine serum albumin at 1 ,g/IAl virus core into the cytoplasm during replication. Thus, host RNAs and 9 ,ug of RNA. This reaction mixture was incubated at other than mRNAs become polyadenylylated in vivo. 37°C for 15 min, and after the addition of 1 ,ul of 10% SDS the RNAs were precipitated in ethanol. MATERIALS AND METHODS RESULTS Cells and Viruses. Human HeLa spinner cells were grown in Eagle's spinner medium supplemented with 5% newborn calf Origin of 5' Ends of POLADS at 1 hr Postinfection (p.i.). serum (NCS). The cells were infected with WR strain of Infection of HeLa spinner cells with VV at a multiplicity of 500 vaccinia virus and purified according to the method of Joklik particles per cell resulted in the inhibition of host protein (11). synthesis as early as 1 hr p.i. and reached a maximum at 3.5 hr Isolation ofTotal Cytoplasmic RNA and poly(A) Containing p.i. POLADS at these two time points were isolated, cloned, RNA from HeLa Cells or W-Infected HeLa Cells. Cytoplasmic and sequenced. Out of 44 pBSPOLADS sequenced, 18 (40%) RNA was isolated according to Krystosek et al. (12) from Abbreviations: POLADS, polyadenylylated RNAs; VV, vaccinia virus; The publication costs of this article were defrayed in part by page charge p.i., postinfection; snRNA, small nuclear RNA; PAB, poly(A)-binding payment. This article must therefore be hereby marked "advertisement" in protein. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2037 Downloaded by guest on September 26, 2021 2038 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996) were from the host, 11 (25%) were from the virus, and 9 had Table 2. Origin of 5' ends of POLADS 1 hr p.i. homologous to no homology to viral or known host sequences and were tRNA probably from the host because the complete sequence of VV Homology, is known (38). Three constructs were too short to be compared Clone Start, nt Stop, nt Origin % to any known sequences, and three others had free poly(A) 64-1 30 75 tRNA-Leu gene-(1-75) 100 stretches of 18-26 nt. These data show that at 1 hr p.i., most 86-1 24 of the 5' ends of POLADS are acquired from the host-cell 64 tRNA-Gly gene-(1-68) 100 RNA. Among 18 cellular POLADS, three were from human small nuclear U2 RNA (snRNA) gene (snRNA-POLADS) shown) indicate that there is no consensus sequence at the (Table 1), two were from tRNA (leucine and glycine) genes 5' ends of POLADS. (tRNA-POLADS) (Table 2), and the remaining 13 5' ends The Effect of Cloned POLADS with Host or Viral 5' Ends originated from various mRNAs (mRNA-POLADS) (Table 3). on Protein Synthesis in Reticulocyte Lysate System. It was Out of the 13 mRNA-POLADS 66% of the sequences do not of interest to test POLADS with host or viral 5' ends for possess the eukaryotic polyadenylylation signal (AAUAAA). inhibitory activity of cellular mRNA translation in the POLADS that contain viral 5' ends are shown in Table 4. cell-free system. A clone of a POLADS homologous to a From 11 viral POLADS, two were from the telomere region portion of bovine casein kinase gene and another homolo- of VV (7.5 K region and NR2 region, respectively), gous to 5' flank of ORF of the VV genome were the remaining nine were from various regions of the VV transcribed in vitro (14). Fig. 1 shows the inhibitory activity genome. of these two POLADS with similar lengths of poly(A) tails The Source of 5' Ends of POLADS at 3.5 hr p.i. Out of a ("80 nt). The results demonstrate that both types of PO- total of 43 POLADS obtained at 3.5 hr p.i., 26 (60%) were LADS inhibit the translation of HeLa mRNAs to a similar homologous to various VV genes, and 10 (23%) were extent (72% and 78% inhibition, lanes 2 and 5). In contrast, homologous to various sequences of the host genome. Four were not found to be homologous to any known sequences, 14( T and three had short 5' ends and could not be evaluated. These data also show that at a relatively late time, 5' ends of 120 POLADS are still derived from both host and viral tran- I 00 scripts. But, unlike at 1 hr p.i., at 3.5 hr p.i. POLADS with c viral 5' ends were more abundant. 80

- Among 10 cellular POLADS isolated at 3.5 hr, two came -;7U 60 from the U2 snRNA gene similar to the U2 RNA POLADS 40. isolated at 1 hr. p.i. The remaining eight were from different region of the host gene (Table 5). Sixty-three percent of these 20

mRNA-POLADS possessed the eukaryotic polyadenylylation i m A. .1 a a I I I II - . . - 4 0) l:--n . .- .i -. -4L itL and a signal, 80% of these POLADS had complete 3' end, 23 4 6 7 8 9 10 1I 12:: -3 14 15 indicating that they may be degradation products of bona fide host mRNAs. Among 26 viral POLADS, 10 (38%) came from the middle region of L2R open reading frame (Table 6), and five (19%) came from the left end of the VV genome (Table 7). The remaining 11 were from various regions of the VV genome (Table 8). 67k- of of Origin 5' Ends POLADS Isolated from Cells Infected 45 o with UV-Irradiated W. UV-irradiated VV does not undergo secondary uncoating (30) and should therefore not release its poly(A) polymerase into the cytoplasm. Cells were in- 30 '- fected with UV-irradiated VV and the POLADS obtained from these were analyzed. The results are shown 20 - in Table 9. Out of 11 UV POLADS, none contained host 5'

ends. This result suggests that nonspecific polyadenylylation C 1 2 3 4 5 6 7 8 9 1{) I 1 12 13 14 15 of host has not occurred probably because the virus cores have not gone through secondary uncoating to release VV FIG. 1. Effect of POLADS with a cellular or viral 5' end on the poly(A) polymerase into the cytoplasm. in vitro translation of HeLa mRNA. POLADS with cellular or viral Consensus Sequence Is Not Required for the Production 5' ends were assayed for their inhibitory activity of HeLa mRNA of POLADS. All POLADS possess 3' poly(A) tails, but their translation in the reticulocyte lysate system. The transcribed PO- 5' ends differ. Thus, it was of interest to determine whether LADS (200 ng) were added to the cell-free translation system. any consensus sequence(s) existed at their 5' ends to act as Mixtures (20 IlI) were subjected to 5-15% PAGE and exposed to an a for a x-ray film. The extent of protein synthesis in each lane from a signal polyadenylylation. Using program (MACAW) representative experiment was determined by laser densitometry. designed to locate and analyze sequence similarities among (Lower) Profile of protein synthesis. Lanes: C, no added RNA; 1, multiple sequences (19-21), we searched for a consensus HeLa total RNA (15 ,ug); 2, HeLa total RNA + H-POLADS; 3, sequence in POLADS. The results of our search (data not HeLa total RNA + H-POLADS + oligo(dT) (2 jig); 4, H-POLADS; 5, HeLa total RNA + V-POLADS; 6, HeLa total RNA + V- Table 1. Origin of 5' ends of POLADS at 1 hr p.i. homologous to POLADS + oligo(dT); 7, V-POLADS; 8, HeLa total RNA + Homo sapiens U2 snRNA gene-(1-187) oligo(dT); 9, viral total RNA; 10, viral total RNA + H-POLADS; 11, viral total RNA + H-POLADS + oligo(dT); 12, viral total RNA Clone Start, nt Stop, nt Homology, % + V-POLADS; 13, viral total RNA + V-POLADS + oligo(dT); 14, 25-1 29 90 96 viral total RNA + oligo(dT); 15, oligo(dT). Numbers at left rep- 69-1 22 90 98.5 resent molecular mass (in kDa) of proteins. (Upper) Magnitude of 83-1 22 88 100 protein synthesis determined by laser densitometry of the autora- diograms. Downloaded by guest on September 26, 2021 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996) 2039 Table 3. Origin of 5' ends of POLADS isolated at 1 hr p.i. homologous to cellular mRNAs Homology, Clone Start, nt Stop, nt Origin N 2a-1* 77 110 H. sapiens cDNA clone HFBDP71-(1-344) 81.8 2b-1* 556,595 556,622 H. sapiens germ-line T-cell receptor (3 chain-(1-684973) 92.8 3-1 1,520 1,700 H. sapiens (subclone H9 3-cll from P1 LBL#3) DNA 83 sequence-(1-3781) 6-1 1,670 1,779 Oryctolagus cuniculus New Zealand White elongation 72.3 factor 1 a (Rabefla2) mRNA, complete cds-(1-1781) 32-1 145 174 H. sapiens cDNA clone b4HB3MA-COT8-HAP-Ftl2l 100 -(1-349) 40-1 1 28 H. sapiens partial cDNA sequence, clone HEA15S-(1-295) 96.4 24 52 96.4 43-1 3,563 3,740 H. sapiens hypoxanthine phosphoribosyltransferase 80.8 (HPRT) gene, complete cds-(1-56737) 54-1 72 172 H. sapiens HepG2 3'-directed Mbo I cDNA, clone 84.1 hmO2dO2-(1-172) 55-1 2 28 H. sapiens HepG2 3'-directed Mbo I cDNA, clone 100 sl4eO2-(1-36) 59-1 1 35 H. sapiens cDNA clone HFBEE41-(1-265) 100 65-1 17,409 17,479 H. sapiens ABL gene, exon lb and intron lb, and putative 78.9 12,671 12,773 M8604 Met protein gene, complete cds-(1-35962) 82 66-1 4 207 H. sapiens cDNA clone b4HB3MA-COT-HAP-Ft227 3', 98 similar to human polyposis locus mRNA-(1-329) 67-1 1 87 H. sapiens expressed sequence TAG-(1-233) 99 88 105 100 76-1 174 227 H. sapiens 8 STS UT5344-(1-426) 83 cds, coding sequence; STS, sequence tagged site. *Clone 2-1 contains an insert region that is homologous to two different regions of the host genome.

both kinds of POLADS had either a slight or no effect on the inhibitory process; rather, the 3'-end poly(A) tails are the translation ofviral mRNAs (3% and 13% inhibition, lanes 10 cause of inhibition. and 12). The inhibition brought about by these POLADS was reversed by the addition of oligo(dT) (lanes 3 and 6). Both POLADS had no translational activity (lanes 4 and 7). DISCUSSION The Effect ofArtificially Constructed POLADS on Protein Synthesis in Reticulocyte Lysate System. POLADS with A relationship between the amount of POLADS produced tRNA, rRNA, or poly(C) at their 5' ends were used to and the extent of inhibition of protein synthesis has been determine whether these normally nonpolyadenylated RNA previously demonstrated (1, 3, 6, 7). To determine the species would inhibit HeLa cell mRNA translation after the derivation of POLADS, we characterized their 5' ends at 1 addition of poly(A) at their 3' ends. The results in Fig. 2 and 3.5 hr after infection. More than half of the POLADS clearly indicate that all three types of POLADS inhibit possessed host cell 5' ends at 1 hr after infection. Because cellular mRNA translation, a situation that mimics the uninfected cells have very limited amounts of small size inhibitory effect of POLADS isolated from infected cells. nontranslated poly(A)+ RNA (10), a mechanism must exist The extent of inhibition by all three POLADS is similar in VV-infected cells that produces POLADS. Among PO- (88%, 94%, and 95% inhibition; lanes 2, 5, and 8, respec- LADS with host cell origin, 17% have 5' ends that are from tively). The poly(A)- portion of these artificially constructed non-mRNA sources including tRNA and small nuclear U2 POLADS does not inhibit HeLa cell mRNA translation RNA. Out of the total sequenced 5' ends derived from host (data not shown). These experiments again demonstrate that mRNAs at 1 hr after infection, 66% have no polyadenylyl- the type of 5' ends of POLADS is not involved in the ation signal, indicating that these host mRNA 5' ends are Table 4. Origin of 5' ends of POLADS isolated at 1 hr p.i. homologous to VV genome Homology, Clone Start, nt Stop, nt Origin % 1-1 827 955 F8L-(817-1014) 98.4 13-1 457 493 virus rpoJ32 gene encoding RNA polymerase-(1-4510) 94.4 14-1 982 1,034 VV B12R gene, complete cds, and B13R gene, 5' flank-(1-1080) 100 18-1 271 330 VV topoisomerase I gene, complete cds-(1-945) 100 30-1 1,274 1,882 VV RNA polymerase subunit gene, complete cds-(1-1928) 98 33-1 183,857 183,932 VV B25R and 5' flank of B25R-(183,882-184,658) 97 183,952 183,992 55 37-1 1,717 1,976 VV L2R-(1698-1961) 99 38-1 906 1,102 VV 13L and 5' flank of 13L-(1099-290) 99 63-1 3,616 3,700 VV genome, left end 96 3,683 3,725 76 73-1 1,111 1,545 VV genome, left end 100 75-1 2 33 VV envelope protein gene-(1-1271) 100 39 103 100 Downloaded by guest on September 26, 2021 2040 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996) Table 5. Origin of 5'-ends of POLADS isolated at 3.5 hr p.i. homologous to cellular genes Homology, Clone Start, nt Stop, nt Origin % 9-3.5 826 880 H. sapiens PROS-27 mRNA-(1-979) 98 869 979 99 13b*-3.5 24 99 H. sapiens U2 snRNA gene-(1-187) 100 14t-3.5 20 159 Human HepG2 3'-directed Mbo I cDNA clone hmOleO6-(1-159) 99 15t-3.5 371 648 H. sapiens adipocyte acid phosphatase mRNA-(1-970) 98 19-3.5 141 343 H. sapiens ribosomal protein S21 (RP21) mRNA gene-(1-343) 98.5 28-3.5 1 22 H. sapiens cDNA clone HRBAA20-(1-211) 100 36-3.5 1 49 IB524 H. sapiens cDNA clone IB524-(1-147) 81.6 46a*-3.5 1 52 H. sapiens cDNA clone HRBAA20-(1-211) 100 67at-3.5 777 845 H. sapiens liver expressed ribosomal protein S3 homologue 98.5 mRNA-(1-845) 81-3.5 29 88 H. sapiens U2 snRNA gene-(1-187) 98.3 *A portion of the insert is also homologous to the virus gene in Table 8. tThe pBS plasmid was used; all others were cloned in pBluescript KS II +/- plasmid. tA portion of the insert is also homologous to the virus gene in Table 6. probably polyadenylylated by the VV poly(A) polymerase. migration of these RNAs to the cytoplasm, where they may act This mode of polyadenylylation seems to be plausible be- as RNA primers to produce POLADS. cause the viral enzyme does not require a specific polyade- Unlike at 1 hr p.i., at 3.5 hr, the percentage of POLADS nylylation signal (22) and is encoded by the viral genome (23, with 5' ends from viral genes is higher (56.8%) than the 24), without apparent cleavage of the RNA substrate (25) percentage of POLADS with 5' ends from host genes and in a biphasic manner (26). Poly(A) polymerase can be (22.7%). This change in the percentage of POLADS is isolated either from purified viral cores or from the cyto- plasm of VV-infected HeLa cells (22, 27). VV poly(A) 12(1 polymerase is released from cores into the cytoplasm during infection, probably after the secondary uncoating, which 10()1I 0 coincided with the beginning of VV DNA replication. VV C So - DNA has been shown to occur within an hour in c replication u cells infected with high multiplicity of VV (28, 29). Thus, c; 60- degraded host RNA with a free 3'-OH may be nonspecifi- cally polyadenylylated by the viral poly(A) polymerase. This -J hypothesis is supported by the finding that POLADS isolated from cells infected with UV-irradiated VV contain only viral 5' ends. Viral cores of UV-irradiated VV do not go through (1 n m I I s I I III I s I secondary uncoating (30), and VV poly(A) polymerase is not 67 I-3... 1 .I E11 I I ",,Asi -:- released into the cytoplasm, resulting only in the polyade- 2 3 4 5 6 7 8 91(111l 12 nylylation of viral RNA primers in the cores. Human U2 snRNA found in the nucleus may also be involved in RNA splicing (31). Changes in the permeability of the nuclear membrane after VV infection may occur and allow 4)5

Table 6. Origin of 5' ends of POLADS isolated at 3.5 hr p.i. homologous to ORF L2R in the Hindlll L fragment-(1-4122) of the VV genome Homology,

Clone Start, nt Stop, nt Origin % 2() . 11-3.5 1955 1996 L2R 95 C I 2 3 4 566 7 8 1) 111 12 1946 1996 100 11*-3.5 1717 1976 L2R 99.6 FIG. 2. Effect of artificial POLADS on the in vitro translation of 1966 1977 100 cellular mRNA. Artificial POLADS (80 ng) made from in vitro 14-3.5 1883 1977 L2R 98.8 polyadenylylation of tRNA, rRNA, or poly(C) were added to the 48-3.5 1721 1977 L2R 99.6 reticulocyte lysate system (25 ,ul) programmed with cellular mR- 51-3.5 1721 1869 L2R 100 NAs. Twenty microliters of the reaction mixture was loaded onto 61-3.5 1944 1978 L2R 100 5-15% PAGE and exposed to x-ray film. The extent of protein 1978 1966 100 synthesis in each lane from a representative experiment was deter- 66-3.5 1721 1980 L2R 99.6 mined by laser densitometry. (Lower) Profile of protein synthesis: 67bt-3.5 1889 1976 L2R 100 lanes: C, system control; 1, mRNA (400 ng); 2, mRNA + tRNA- 78-3.5 1940 1971 L2R 90 POLADS; 3, mRNA + tRNA-POLADS + PAB (8 jig); 4, mRNA 82-3.5 1699 L2R 100 + tRNA-POLADS + oligo(dT) (2 ,ug); 5, mRNA + rRNA- 1977 POLADS; 6, mRNA + rRNA-POLADS + PAB; 7, mRNA + ORF, open reading frame. rRNA-POLADS + oligo(dT); 8, mRNA + poly(c)-POLADS; 9, *The pBS plasmid was used; all others were cloned in pBluescript II mRNA + poly(c)-POLADS + PAB; 10, mRNA + poly(c)-POLADS KS +/- plasmid. + oligo(dT); 11, HeLa mRNA + PAB; 12, HeLa mRNA + tA portion of the insert is also homologous to the host gene in Table oligo(dT). (Upper) Magnitude of protein synthesis determined by 5. laser densitometry of autoradiograms. Downloaded by guest on September 26, 2021 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996) 2041 Table 7. Origin of 5' ends of POLADS at 3.5 hr p.i. homologous Table 9. Identification and genome localization of POLADS from to the left end of the VV genome-(1-14,520) UV-irradiated W-infected cells Homology, Homology, Clone Start, nt Stop, nt Origin % Clone Start, nt Stop, nt Origin % 7*-3.5 3603 3872 7.5-kDa region 100 UVi 7,495 7,597 Left end 99.0 18-3.5 3629 3695 7.5-kDa region 92.5 UV4 555 614 19K 100 53-3.5 1102 1174 NR2 region 100 UV5 81,072 81,194 L3L 100 77-3.5 3624 3848 7.5-kDa region 95.5 UV6 7,686 7,745 Left end 99.0 80-3.5 3625 3678 7.5-kDa region 100 UV1l 68,477 68,677 16L 99.0 *The pBS plasmid was used; all others were cloned in pBluescript II UV12 1,633 1,690 Ml and N2 genes 98.2 KS +/- plasmid. UV18 20,109 20,324 CSL 99.0 UV21 29,078 29,207 KlL 99.1 probably due to increased viral transcription and translation UV23 173,985 174,124 B1SR 99.0 at later times, which would increase the probability for the production of POLADS with viral 5' ends. Among POLADS rRNA fragments would nonspecifically bind to oligo(dT)- isolated at 3.5 hr p.i., two cellular POLADS were also found cellulose during the poly(A)+ RNA isolation. We found, with human snRNA 5' ends. This finding suggests that by Northern blot analysis, that this is the case (data not nonspecific polyadenylylation by VV poly(A) polymerase shown). Therefore, it is possible that during cDNA synthesis, occurs continuously. these rRNAs may form a secondary structure including a L2R is an open reading frame that is conserved in both loop structure which may act as a primer to start cDNA fowlpox and variola virus (32), but its biological function is not synthesis. yet known. Our results show that the L2R open reading frame In addition to the characterization of POLADS, this study is an early or early/late gene because 5' ends of POLADS from has also added to our knowledge of VV replication. One of this region are found both at 1 hr and 3.5 hr. The highest the POLADS has a 5' end from the NR2 region and the expression of this gene occurs -1.5 hr p.i. (data not shown). adjacent repeat sequence. This sequence contains an Alu I At 3.5 hr, the expression of POLADS from the 7.5 kDa region site, which is a unique site in the NR2 region, and a Hinfl site, also increases. The reason why most viral POLADS come from which is a site that occurs in the repetitive sequences (34). these two regions at 3.5 hr is unclear; it may be due to the high Parsons and Pickup (35) demonstrated that this noncoding error expression rate of these genes or the rapid degradation sequence was transcribed at the late time after infection. We of their mRNAs. find that this noncoding region is also expressed as a Because there is a diversity among 5' ends of POLADS, we POLADS at 1 hr p.i. Clone 18-1 contains a sequence that tried to determine whether there was any consensus sequence comes from VV topoisomerase I gene (H6R) (36). We find for polyadenylylation. VV poly(A) polymerase does not need that even though this open reading frame has a late tran- specific sequences for polyadenylylation; however, uridylate- scriptional signal (37), it is expressed at 1 hr p.i. The function containing RNA >31-40 nt in length are necessary for both and time of expression of open reading frame F8L and open binding of VV poly(A) polymerase and polyadenylylation of reading frame B25R are not known. We find that both these mRNAs (33). We also find no consensus sequence among genes are expressed at early times after infection. these isolated POLADS. Thus, it can be concluded that VV According to these results, a general model of inhibition of poly(A) polymerase can polyadenylylate any in vivo RNA host protein synthesis by VV is proposed. VV has evolved to sequence without a specific signal. produce POLADS in infected cells by means of its promiscu- The results shown in Figs. 1 and 2 demonstrate that all types ous poly(A) polymerase, which can adenylate any RNA primer of POLADS inhibit HeLa mRNA translation equally well in without sequence specificity. In this fashion, VV infection the reticulocyte lysate system. The inhibition caused by these leads to an increase in the level of poly(A) in the cytoplasm, various types of POLADS can also be reversed by the addition which sequesters poly(A)-binding protein (PAB), an essential of oligo(dT), indicating that the inhibitory property resides in initiation factor. This sequestration of PAB selectively inhibits their poly(A) tails. host mRNA translation because viral mRNAs can still initiate Five of our clones also contained cDNA inserts from the under limiting amounts of PAB (5). Whether mechanisms 28S rRNA gene. However, none of these inserts contained a other than POLAD production also contribute to regulation of poly(A) tail at their 3' ends. Thus, these inserts are not translation and shut-off during VV infection remains to be considered to be POLADS. We tested to see whether 28S elucidated. Table 8. Origin of 5' ends of POLADS isolated at 3.5 hr p.i. homologous to the VV genome Homology, Clone Start, nt Stop, nt Origin % 1*-3.5 1,527 1,807 A44L-(1811-1493) 94.6 10-3.5 45 102 VV envelope protein gene-(1-1271) 100 13at-3.5 1,731 1,814 VV abortive gene-(1-2955) 100 15-3.5 105,770 105,850 D7R-(105,758-106,243) 95 21-3.5 25,550 25,311 N2L-(25,886-25,240) 100 23-3.5 1,408 1,443 5' flank of F6L-(1420-1616) 100 27-3.5 984 1,033 5' flank of B13R 100 44-3.5 916 940 VV cells surface-binding protein gene-(1-1020) 96 916 966 46bt-3.5 1,840 1,869 VV RNA polymerase subunit gene-(1-1928) 100 49-3.5 7,762 7,872 C19L-(7855-7080) 96.7 72-3.5 1,425 1,634 VV DNA-dependent RNA polymerase subunit (Rpo 19) gene-(1-3150) 95.7 *The pBS plasmid was used; all others were cloned in pBluescript II KS +/- plasmid. tA portion of the insert is also homologous to the host gene in Table 5. Downloaded by guest on September 26, 2021 2042 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996)

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