Transcription and Replication Initiate at Separate Sites on the Vesicular Stomatitis Virus Genome

Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome

Sean P. J. Whelan* and Gail W. Wertz†

Department of Microbiology, University of Alabama School of Medicine, 845 19th Street S, Birmingham, AL 35294

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved May 28, 2002 (received for review March 15, 2002) The RNA-dependent RNA polymerase of the nonsegmented negative- Studies on a VSV mutant, polR1, with a single amino acid change strand RNA viruses carries out two distinct RNA synthetic processes: in the template-associated N protein, provided evidence that con- transcription of monocistronic, capped, and polyadenylated sub- flicts with the single-initiation site model (16). PolR1 virus synthe- genomic messenger RNAs, and replication by means of the synthesis sized N mRNA in excess over Leϩ, which suggested a two-entry site of a full-length positive-sense copy of the genome. The template for model for RNA synthesis: 3Ј entry for synthesis of Leϩ and the both processes is the negative-sense genomic RNA tightly encapsi- full-length antigenome, and direct entry at the first gene-start site dated by the viral nucleocapsid protein. By applying UV transcrip- for synthesis of mRNAs (17). tional mapping to engineered variants of vesicular stomatitis virus, Reverse genetic systems developed for the nonsegmented neg- we discovered that, in infected cells, transcription and replication are ative-sense (NNS) RNA viruses (18, 19) led to identification of controlled by initiation at different positions on the viral genome. signals that regulate gene expression. Analysis of the VSV intergenic junction defined signals for mRNA termination and initiation he genome of vesicular stomatitis virus (VSV), the prototypic and provided support for the stop-start model (20–27). Analysis of Trhabdovirus, comprises 11,161 nucleotides of negative-sense the VSV genomic termini identified sequences essential for tran- RNA. This RNA, tightly encapsidated by the viral nucleocapsid (N) scription and replication (28–33). However, these studies were protein, forms the template for the RNA-dependent RNA poly- unable to address the stop-start model of sequential transcription at merase (RdRP), the viral components being a 241-kDa large the Le-N gene junction. subunit (L) and a phosphoprotein (P) (1, 2). The RdRP uses the Determination of the polymerase initiation site is pivotal to genome as a template for two reactions: (i) transcription of a short understand global regulation of RNA synthesis in VSV, and by leader RNA (Leϩ) and 5 mRNAs that encode N, P, matrix (M) extrapolation other NNS viruses. If transcription and replication ϩ protein, glycoprotein (G), and L; and (ii) replication to yield initiate at nucleotide 1, then during synthesis of Le a regulatory full-length antigenomic and then genomic strands. event must occur to determine whether polymerase transcribes or The VSV gene order was determined by transcriptional mapping replicates the genome. This regulatory event was postulated to be ϩ using ultraviolet (UV) radiation. Low-dose UV radiation induces encapsidation of nascent Le with N protein (34, 35). Conversely, formation of covalent dimers between adjacent thymine (T) or if transcription and replication initiate at separate positions, regu- uracil (U) residues in nucleic acids (3–5). These block progression lation must occur before synthesis. Ј of polymerase during transcription and thus show the distance We examined whether polymerase initiates transcription at the 3 polymerase traverses to transcribe a gene. Such experiments de- end of the genome, or directly at the first gene start. Recombinant termined the VSV gene order as 3Ј-N-P-M-G-L-5Ј and showed the viruses were generated to test this question by using the technique mRNAs were synthesized in an obligatory sequential manner after of UV-mapping. These viruses contained an additional small gene polymerase entry at a single 3Ј promoter (6–8). The abundance of (I), either 60 or 108 nt, inserted just downstream of the leader viral mRNAs was shown to decrease with distance from the region. In addition, the sequence of the leader region was altered promoter such that N Ͼ P Ͼ M Ͼ G Ͼ L (9). This relative to increase or decrease the number of adjacent U residues, such that ϩ abundance reflected a localized transcriptional attenuation, the UV sensitivity of Le would be altered in a predictable manner. ϩ whereby 30% of polymerase molecules reaching each gene junction The effect of these changes on the UV sensitivity of Le and the failed to transcribe the downstream gene (10). I mRNA was examined in vitro and in infected cells. These The accepted model for VSV transcription is the stop-start experiments confirmed that transcription of the first gene required model, which posits that polymerase enters the genome at a single prior transcription through the leader region in vitro. In infected site and that transcription of a downstream gene depends on cells, however, we demonstrate that polymerase initiates transcrip- termination of transcription of the upstream gene (11). The N gene tion directly at the first gene start sequence. is preceded by a 50-nt leader region, which is transcribed into a 47-nt Materials and Methods RNA (Leϩ) (12–14). This leader region is too small to contribute to the UV sensitivity of the 1,336-nt N mRNA. Thus UV mapping Recombinant Viruses. An infectious cDNA clone of VSV, ϩ studies could not determine whether transcription of N required pVSV1( ) (36), was modified by standard methods (37). The I Ј prior transcription of Leϩ. gene sequences were 3 -UUGUCAUUAGUCUUAAGAGCU- In vitro reconstitution studies suggested a single polymerase entry CUACCGUAUGACAUUGGUAUUCCGGUAUACUUU- Ј Ј site. In these experiments, polymerase was added to template in the UUUUGA-5 (I-60), and 3 -UUGUCAUUAGUCUUAA- presence of limiting nucleoside triphosphates (NTPs), and the GAGCUCGUGUUGGUUGGUCUUGCACUUUUUCGC- products of de novo synthesis were analyzed (15). In the presence AGGACGCACAUCGCUUGACGCUACCCGUAUGACA- of ATP and CTP, the major product was 5Ј-pppApC-3Ј, represent- ing the first 2 nucleotides of Leϩ. The tetranucleotide 5Ј- pppApApCpA-3Ј, corresponding to the first 4 nucleotides of the N This paper was submitted directly (Track II) to the PNAS ofﬁce. mRNA, was found only in a two-step reaction. In this reaction, all Abbreviations: VSV, vesicular stomatitis virus; RdRP, RNA-dependent RNA polymerase; N, nucleocapsid; L, large subunit; P, phosphoprotein; M, matrix; G, glycoprotein; Leϩ, leader NTPs were present, the reaction was stopped, NTPs were removed, RNA; NNS, nonsegmented negative-sense; WT, wild type; pfu, plaque-forming units; Inc-U, and the products of synthesis in the presence of ATP and CTP were increased-U leader; Dec-U, decreased-U leader. analyzed. These experiments indicated that the RdRP could access *Present address: Department of Microbiology and Molecular Genetics, Harvard Medical the template only at the extreme 3Ј end, and that transcription of School, 200 Longwood Avenue, Boston, MA 02115. N required prior transcription of Leϩ. †To whom reprint requests should be addressed. E-mail: gail࿝[email protected].

9178–9183 ͉ PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.152155599 Downloaded by guest on September 28, 2021 UUGGUAUUCCGGUAUCUUUUUUUGA-5Ј (I-108). Po- tential sites for UV-induced U dimers are underlined. Viruses were recovered, amplified, and purified as described (36). Se- quences were confirmed by reverse transcription–PCR on genomic RNA by using a primer that annealed to the first 15 nt of the genome and a second primer designed to anneal to the complement of nucleotides 145–166 in the N gene of the wild-type (WT) genome.

UV Irradiation. Purified virus was diluted in PBS to 2 ϫ 109 plaque-forming units (pfu)͞ml. Aliquots (0.25 ml) were dispensed into 6 wells of a 24-well plate on ice, exposed to UV radiation at 31 erg⅐mmϪ2⅐sϪ1 for 0–8 min at a distance of 54 cm from a 30-W UV bulb. Irradiations were performed in three groups: (i) rV1ϩ60 WT, rV1ϩ60incU, and rV1ϩ60decU for in vitro analysis; (ii)rV1ϩ60 WT, rV1ϩ60incU, and rV1ϩ60decU for in vivo analysis; and (iii) rV1ϩ60 and rV1ϩ108 for in vivo analysis. Fig. 1. Effect of UV radiation on VSV mRNA synthesis in infected cells. (A) Schematic of VSV with an additional gene between leader and the N gene and Analysis of RNA Synthesis in Infected Cells. Before infection, baby Ј Ј ␮ ͞ alterations to the leader sequence. The negative-sense genome is shown 3 -5 . hamster kidney (BHK-21) cells were treated with 10 g ml cyclo- The sequences of the leader regions are shown. le, leader; tr, trailer; Inc-U, heximide (CHX) and 10 ␮g͞ml actinomycin-D (act-D) for 15 min. increased-U leader; Dec-U, decreased-U leader. Note positions of nucleotide Cells were infected with VSV at a preirradiation multiplicity of identity are indicated by a -. (B) Effect of UV radiation on mRNA synthesis. infection of 100, in duplicate. Virus was adsorbed for 45 min and Purified viruses were exposed to UV radiation before infection of BHK cells. cells were exposed to [3H]uridine in the presence of act-D and CHX RNAs were labeled, extracted, treated with RNase H before electrophoresis, from 0.25 to 5.25 h after infection. Cells were harvested, cytoplas- and visualized by fluorography. The mobility of the individual mRNA species mic extracts were prepared, and RNAs were analyzed on agarose is indicated (L, G, N, P, M). Note: (i) the P and M mRNAs comigrate; and (ii) the small size and low U content of the I mRNA make its detection difficult by urea gels (18, 31). direct metabolic labeling. BIOCHEMISTRY VSV RNA Synthesis in Vitro. No quantitative difference in the UV sensitivity of the I mRNA was obtained when VSV was detergent comprised the gene-start (3Ј-UUGUCNNUAG-5Ј), 37 nt of stuffer disrupted or intact before UV irradiation (not shown). In vitro sequence, and the gene-end and intergenic dinucleotide (3Ј- transcriptions used detergent-activated virions (38). Purified virus AUACUUUUUUUGA-5Ј). In addition, two other viruses were ͞ activated with 0.05% Triton N-101 0.4 M NaCl (5 min, 25°C) was generated that altered the number of UV target sites in the leader ⅐ diluted with 10 mM Tris HCl, pH 8.1, before irradiation. Reaction region (Fig. 1A and Table 1). The rationale was that if transcription ϫ 8 ␮ mixtures contained 1 10 pfu of virus in 100 l that contained 30 initiated at the 3Ј end of the genome, these alterations would affect ␮ ␮ l of nuclease-treated rabbit reticulocyte lysate (Promega), 10 lof the UV sensitivity of the I-60 mRNA. Conversely, if the RdRP ϫ ⅐ ͞ ͞ 10 buffer (0.3 M Tris HCl,pH8.10.33 M NH4Cl 0.07 M bypassed the leader region and instead initiated transcription ͞ ͞ ͞ ͞ KCl 0.045 M Mg(OAc)2 0.01 M DTT 0.002 M spermidine 10 directly at the first gene-start, these changes would not affect the ͞ ␮ mM ATP 5 mM each GTP, CTP, and UTP), and 5 lof UV sensitivity of I-60. Previously we identified sequences in the ͞ actinomycin-D (1 mg ml). Duplicate reactions were incubated at leader region essential for transcription and replication (33). 30°Cfor5h. Guided by these analyses, we generated two variants of rV1ϩ60: one in which the number of adjacent U residues in leader was ϩ Primer Extension Analysis. The I mRNAs and Le RNAs were increased (rV1ϩ60incU) and a second in which they were de- detected by primer extension, using specific oligonucleotide primers creased (rV1ϩ60decU) (Fig. 1A). The sequence of the leader extended by Superscript II reverse transcriptase (Invitrogen) at region of these viruses was confirmed by reverse transcription–PCR Ј 50°C. The I mRNA was detected by primer (5 -TTTTTTTTTTT- (data not shown). All viruses grew to similar titers (Ͼ109 pfu͞ml) Ј TCATA-3 ) designed to anneal to all VSV mRNAs. Leader RNAs and had similar plaque sizes, indicating that viral growth was not were detected by using primers designed to anneal to nucleotides measurably affected (data not shown). ϩ Ј Ј ϩ 47–33 of WT Le (5 -GTTTCTCCTGAGCC-3 ), Inc-U Le The effect of these alterations on viral mRNA synthesis and on Ј Ј ϩ Ј (5 -TTTTTTCATAGGCC-3 ), and Dec-U Le (5 -GTCTCTC- the UV sensitivity of the N, P, M, G, and L mRNAs was examined. CTGAGCC-3Ј). Primers were end-labeled by using [␥-33P]ATP and T4 polynucleotide kinase (Invitrogen). Labeled primers were purified from unincorporated [␥-33P]ATP by using a QIAquick Table 1. Number of U dimers that can form on UV irradiation nucleotide removal kit (Qiagen). Labeled primer (0.2 pmol) was Maximum no. of U dimers annealed with 1͞25 of the total RNA from a 60-mm dish or an in vitro transcription reaction. Products were analyzed by electro- I-60 I-108 phoresis on denaturing 6% polyacrylamide gels and quantitated by Virus Leader 3Ј Internal 3Ј Internal densitometric scanning (39). Dec-U 3 11 8 Results WT 6 14 8 20 14 VSV Engineered to Contain an Additional Gene Inserted at the Inc-U 8 16 8 Leader–N Gene Junction and Modifications to the Sequence of the Leader Region. To examine whether polymerase initiates transcrip- The maximum number of covalent U dimers that could form on UV irradiation are shown. For I-60 and I-108 the values are shown for initiation of tion directly at the first gene-start sequence or must first transcribe Ј Ј ϩ synthesis at the 3 end (3 ) or for initiation of synthesis directly at the first gene Le , we generated recombinant viruses that allowed UV mapping start site (Internal). Sequences are shown in Fig. 1A and Materials and Meth- to discriminate these two possibilities. We used a recombinant VSV ods. To calculate the number of U dimers we used the following assumptions: (rV1ϩ60) that contained an additional 60-nt gene (I) inserted U3 can only form 1, U4 and U5 can form a maximum of 2, and U6 and U7 can between the leader region and the N gene (39). This additional gene form a maximum of 3.

Whelan and Wertz PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9179 Downloaded by guest on September 28, 2021 Fig. 2. Effect of alterations to the leader region on the UV sensitivity of the Fig. 3. Effect of alterations to the leader region on the UV sensitivity of the I leader RNA synthesized in vitro.(A) Primer extension analysis of the leader RNAs. mRNA synthesized in vitro.(A) Primer extension analysis of the I mRNA. Reverse Reverse transcriptions were performed with primers designed to anneal to po- transcriptions were performed and the products were analyzed. (B) Quantitation ■ sitions 33–47 of the individual leader RNAs. Products were analyzed on denatur- of the I-60 mRNAs. The autoradiographs in A were scanned and analyzed. ,WT F Œ ing 6% polyacrylamide gels, and the individual leader RNAs are shown (Le). (B) I-60; , Inc-U I-60; , Dec-U I-60. Quantitation of the leader RNAs. The autoradiographs in A were scanned and analyzed. For each Leϩ the amount remaining was plotted as the percentage of synthesis directed by the unirradiated control. ■, WT; F, Inc-U; Œ, Dec-U. doses of UV radiation was expressed as a percentage of the average value obtained from the unirradiated control, and was plotted (Fig. 2B). The rate of decrease of synthesis for each Leϩ was linear as Viruses were resuspended in PBS at a titer of 2 ϫ 109 pfu͞ml and demonstrated by the lines of best fit. This result indicated that Ϫ Ϫ exposed to UV radiation at a dose of 31 erg⅐mm 2⅐s 1 for 0–2min. inhibition of synthesis followed single-hit kinetics. According to the Duplicate confluent monolayers of BHK cells were infected with Poisson distribution, each member of the population receives, on VSV at a preirradiation titer of 1 ϫ 108 pfu. Cells were pretreated average, a single UV hit when the abundance of the RNA is with actinomycin-D to inhibit DNA-dependent RNA synthesis, and reduced to 37%. This 37% survival dose was determined as 39,060, cycloheximide to prevent protein synthesis and thus prevent repli- 20,460, and 12,090 erg⅐mmϪ2 for the Leϩ synthesized by Dec-U, cation of undamaged genomes. This treatment ensured that the WT, and Inc-U, respectively. Thus the UV sensitivity of Leϩ only viral RNA synthesis that occurred was primary transcription. depended on the number of adjacent U residues in the leader Viral RNAs were labeled, extracted, annealed with oligo(dT) region. The maximum number of U dimers that could form in each before exposure to RNase H to remove polyadenylate tails, ana- template was 3, 6, and 8 for Dec-U, WT, and Inc-U, respectively lyzed by electrophoresis, and visualized by fluorography (Fig. 1B). (Table 1). These numbers predict relative survival doses of 1.00, The sensitivity of the viral mRNAs to UV radiation were similar for 0.50, and 0.375 for Dec-U, WT, and Inc-U, in good agreement with rV1ϩ60, rV1ϩ60decU, and rV1ϩ60incU in that L Ͼ G Ͼ M Ͼ P Ͼ the measured values of 1.0, 0.5, and 0.3. These data thus show that N. This result demonstrated that the alterations to the viral genome altering the number of adjacent U residues in the leader region had not altered transcription, and confirmed the sequential nature affected the UV sensitivity of Leϩ in a predictable manner. of transcription described for WT VSV (6–8). Because of their size, the contribution of the leader region and the 60-nt gene to the UV Effect of UV Radiation on mRNA Synthesis in Vitro. To determine sensitivity of the N, P, M, G, and L mRNAs was undetectable. In whether transcription of the first gene required transcription addition, the low U content of the I-60 mRNA made it difficult to through the leader region, we examined the UV sensitivity of the detect this species by metabolic labeling. I-60 mRNA synthesized in vitro. The I-60 mRNA was detected by primer extension using oligonucleotide primer 5Ј- Effect of Altering the U Content of the Leader Region on the UV TTTTTTTTTTTTCATA-3Ј (Fig. 3A). This primer annealed to Sensitivity of Leader RNA. To examine the effect of alterations to the each VSV mRNA by virtue of their common 3Ј-terminal sequence ϩ Ј U content of the leader region on the UV sensitivity of Le , VSV 5 . . . UAUG(A)n. The products of extension on the viral N, P, M, transcription was analyzed in vitro. Detergent-disrupted virus was G, and L mRNAs would be large (Ͼ800 nt), and are therefore not exposed to UV radiation at a dose of 31 erg⅐mmϪ2⅐sϪ1 for 0–8min. visualized. For each virus, the product of primer extension on the In vitro transcriptions were performed in duplicate and RNAs were I-60 mRNA was detected, and its abundance diminished with recovered. The abundance of each Leϩ was determined by primer increasing doses of UV radiation (Fig. 3A). The I-60 mRNA extension (Fig. 2A). For each virus, the 47-nt product of primer abundance was determined and plotted (Fig. 3B). The rate of extension on the Leϩ was detected, and its abundance diminished decrease of synthesis of I-60 was linear as demonstrated by the lines as the dose of UV radiation increased. Reactions were performed of best fit, confirming single-hit kinetics. The 37% survival dose for under conditions of primer excess to ensure quantitative detection. I-60 synthesized by Dec-U, WT, and Inc-U was determined as Leader RNA abundance was determined by densitometric scanning 11,720, 9,110, and 6,880 erg⅐mmϪ2, respectively. Thus, altering the of the autoradiographs. The amount of Leϩ remaining after varied number of adjacent U residues in the leader region affected the UV

9180 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.152155599 Whelan and Wertz Downloaded by guest on September 28, 2021 Table 2. Fitted regression analysis for UV sensitivities of RNA synthesized in infected cells 37% survival dose, RNA Slope R2,% erg⅐mmϪ2

Inc-U (Leϩ) Ϫ0.371 Ϯ 0.028 99.3 5,000 Dec-U (I-60) Ϫ0.352 Ϯ 0.030 98.7 5,250 WT (I-60) Ϫ0.360 Ϯ 0.028 99.0 5,130 Inc-U (I-60) Ϫ0.441 Ϯ 0.022 99.6 4,190

Regression analysis was performed on the data generated from densitometric scanning (Fig. 4A). These values were expressed as percentages (Fig. 4B). The fitted regression lines are of the form ln(y) ϭ intercept ϩ slope(exposure time). The R2 value is a measure of how tightly the data values fit about the regression line with maximum possible value of 100%. Values for the slopes are given within 95% confidence limits.

entry and transcription of Leϩ before I-60, the maximum numbers of U dimers that could form in each template were 8, 11, 14, and 16 for Inc-U (Leϩ), Dec-U (I-60), WT (I-60), and Inc-U (I-60), respectively (Table 1). This finding yields a ratio of predicted survival doses of 1.00:0.73:0.57:0.50. In contrast, the measured ⅐ Ϫ2 Fig. 4. Effect of alterations to the leader region on the UV sensitivity of the I values 5,000, 5,250, 5,130, and 4,190 erg mm (Table 2) yielded relative sensitivities of 1.00:1.05:1.03:0.84. These data are inconsis- mRNA synthesized in infected cells. (A) Primer extension analysis of the I mRNA. Ј Reverse transcriptions were performed and the products were analyzed. Note tent with 3 entry of polymerase and sequential transcription of that because of sequence identity at the end of the leader RNA, the primer Leϩ before I-60. Rather they indicate that transcription initiated anneals to the leader synthesized from rV1ϩ60incU. This product is shown (Le). directly at the first gene-start sequence, which predicts a ratio of

(B) Quantitation of the I mRNAs. The autoradiographs in A were scanned and relative sensitivities of 1.0:1.0:1.0:1.0. A separate slopes linear BIOCHEMISTRY analyzed. ■, WT I-60; F, Inc-U I-60; Œ, Dec-U I-60; ᭜, Inc-U Leϩ. Two values for each regression analysis (40) comparing all lines was performed. This time point were plotted to generate the graphs shown. Because of the tight analysis indicated that the slopes for Inc-U (Leϩ), Dec-U (I-60), distribution of the data points, many points are superimposed on one another. and WT (I-60) were not significantly different from one another Statistical analysis of the data is presented in Table 2. Dashed line represents (P Ͼ 0.3). The slope for Inc-U (I-60) was slightly less than the other the predicted UV sensitivity for Inc-U (I-60) if transcription were obligatorily Ͻ sequential. three slopes (P 0.001). However, this difference was not compatible with obligatorily sequential transcription of leader RNA before synthesis of the I-60 mRNA. For these data to support sensitivity of I-60. The maximum numbers of U dimers that can obligatorily sequential transcription of Leϩ before I-60, the slope form in each template are 11, 14, and 16 for Dec-U, WT, and Inc-U, of the Inc-U (I-60) would need to differ 2-fold from the existing respectively (Table 1). Thus the predicted relative sensitivity of I-60 Inc-U (Leϩ). This difference would result in the hypothetical was 1.00, 0.79, and 0.69 for Dec-U, WT, and Inc-U, respectively. dashed line for Inc-U (I-60) instead of the experimentally deter- This result agrees with the ratio of experimentally derived values of mined solid line (Fig. 4B). Consequently these data show that 11,720:9,110:6,880 or 1.0:0.8:0.6. These data showed that altering polymerase initiates transcription directly at the first gene-start site the number of adjacent U residues in the leader region affected the in infected cells. These findings were confirmed by multiple inde- UV sensitivity of the I-60 in a predictable manner. This observation pendent experiments that compared the relative sensitivity of the is consistent with 3Ј entry and obligatorily sequential transcription Inc-U (I-60) and Dec-U (I-60) by using a different primer (data not of Leϩ before synthesis of I-60, and not with direct initiation of shown), and by comparing the sensitivity of I-60 with a larger transcription at the first gene-start. Thus we confirmed previous mRNA (I-108) as described below. work demonstrating that transcription initiates at the 3Ј end of the genome in vitro (15). These data also extended the utility of UV Sensitivity of the First Gene Is Altered by Its Base Composition. The UV-mapping experiments by demonstrating that the U content of above experiments indicated that in infected cells, polymerase an RNA genome can be manipulated to affect the UV sensitivity bypassed the leader region and instead initiated transcription of a downstream transcript in a predictable manner. directly at the first gene-start. To confirm this, we generated an additional recombinant VSV (rV1ϩ108) that contained a 108-nt Effect of UV Radiation on I mRNA Synthesis in Infected Cells. The gene (Table 1) inserted between the WT leader region and the N experiments described above showed that transcription initiated at gene and compared the relative UV sensitivity of I-60 and I-108. nucleotide 1 with synthesis of Leϩ before transcription of the first Purified viruses were exposed to UV radiation for 0–5 min before gene in vitro. To examine this issue in vivo we determined the UV infection of cells. RNAs were extracted, and the I mRNA was sensitivity of I-60 synthesized by each virus in infected BHK cells. detected by primer extension. Synthesis of I mRNA decreased as The I-60 mRNA was detected by primer extension analysis (Fig. 4). the extent of UV irradiation increased (Fig. 5A). The abundance of For rV1ϩ60incU, the primer detected leader RNA in addition to the I mRNAs was determined and plotted to obtain the graph the I-60 mRNA, because the sequence at the end of the leader shown in Fig. 5B. The 37% survival doses were 3,000 erg⅐mmϪ2 for region of this virus was identical to that found at the end of each I-108 and 5,400 erg⅐mmϪ2 for I-60, a 1.8-fold difference in relative VSV gene (3Ј-AUACUUUUUUUGA-5Ј). The abundance of I-60 sensitivity. The predicted relative sensitivity of the I-60 and I-108 and the Inc-U Leϩ was determined (Fig. 4B). For each RNA, the mRNA are 1:1.43 (14:20) for polymerase that initiates transcription rate of decrease of synthesis was linear as shown by the respective at the 3Ј end of the genome, or 1:1.75 (8:14) for polymerase that lines of best fit; thus inhibition of synthesis followed single-hit initiates directly at the first gene-start. Consequently, the measured kinetics. In contrast to the in vitro analysis described above, each of 1.8-fold difference is compatible with polymerase entry at the first the lines of best fit was tightly clustered. Linear regression analysis gene-start sequence rather than at the extreme 3Ј end of the was used to determine the 37% survival doses (Table 2). For 3Ј genome.

Whelan and Wertz PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9181 Downloaded by guest on September 28, 2021 Fig. 6. Model for RNA synthesis: Schematic of the VSV genome depicting the leader region (Le) and the N and P genes. During transcription the RdRP, a complex of L (large oval) and a trimer of P (small oval), binds to speciﬁc sequences and initiates synthesis at the N gene start. The products of this reaction are the VSV mRNAs, of which the N and P mRNAs are shown. The 5Ј-terminal cap is depicted by a black diamond, and the 3Ј polyadenylate tail by A(n). During replication, the RdRP initiates at the 3Ј end of the genome. Initiation at the 3Ј end provides leader RNA (not shown) and the full-length antigenome. Replication requires protein synthesis to supply N protein for encapsidation of nascent RNA. N protein (hatched oval) is kept soluble by interaction with P, in a 2:1 complex. Positions of initiation are indicated by the black triangles. Fig. 5. Effect of UV radiation on the synthesis of the I mRNA in infected cells. (A) Primer extension analysis of the I mRNA. Reverse transcriptions were performed and the products were analyzed. The I mRNAs are indicated I-60 and I-108. genome at a single site and initiates transcription at position 1, (B) Quantitation of the I-60 and I-108 mRNAs. The autoradiograph in A was synthesizing Leϩ. Termination of Leϩ is essential to allow poly- ■ Œ scanned and analyzed. , I-60; , I-108. merase to transcribe the N mRNA. In turn, termination of the N mRNA is essential for transcription of P, and so on. The experi- Discussion ments described here show that polymerase initiates transcription directly at the first gene-start in infected cells, a finding that is We used UV transcriptional mapping to examine whether the VSV Ј incompatible with the stop-start model applying at the leader–N RdRP initiated transcription at the 3 end of the genome, or directly gene junction. Our results do not suggest that polymerase enters at the first gene-start site. We inserted a 60-nt gene (I) immediately internally at the other gene-starts on the genome as was initially downstream of the leader region, and altered the number of proposed in a multiple entry site model. Such a model is incom- potential UV target sites in the leader region by changing the patible with the original UV-mapping experiments (6–8) and the number of adjacent U residues. Analysis of RNAs synthesized in ϩ data shown in Fig. 1. Rather, we propose a modification to the vitro showed that the UV sensitivity of Le was proportional to the stop-start model of sequential transcription (Fig. 6). We suggest maximum number of U dimers that can form on exposure to UV that during transcription, polymerase is recruited to the template radiation. These in vitro experiments also showed that the UV through recognition of specific sequence elements that include the sensitivity of the I mRNA was affected by these alterations to the previously defined RdRP-binding site, and probably other sequence leader region, confirming earlier in vitro studies that showed Ј elements identified as essential for mRNA synthesis (29, 33). transcription initiates at the 3 end of the genome with obligatorily Transcription initiates directly at the N gene-start, possibly by direct ϩ sequential synthesis of Le before transcription of the first gene. positioning of the polymerase active site. During replication, poly- In contrast, analysis in infected cells provided evidence that was merase is recruited to the 3Ј end of the genome through recognition Ј incompatible with 3 entry and obligatorily sequential synthesis of of specific sequence elements that may overlap those defined for ϩ Le before transcription of the first gene. Rather, the in vivo transcription. However, in this case the active site is positioned so experiments showed that the RdRP initiated transcription directly synthesis initiates at nucleotide 1. Alternatively, all polymerase at the first gene-start sequence. Three lines of evidence supported could enter the genome at position 1, and during transcription scan these conclusions: (i) the UV sensitivity of I-60 mRNA synthesized through the leader region to initiate transcription at the first in vivo,(ii) the demonstration that the I-60 mRNAs and the Inc-U gene-start. Leϩ RNA had similar UV sensitivities, and (iii) the relative sensitivities of the 60- and 108-nt genes. These data cannot elimi- Separate Initiation Sites for Transcription and Replication. A central nate the possibility that some RdRP initiates transcription at the 3Ј unresolved aspect of the molecular biology of NNS viruses is how end of the genome in infected cells; however, these data show that transcription and replication are coordinately regulated. Immedi- the RdRP predominantly initiates transcription directly at the first ately on infection of a cell, viral genomes are transcribed by the gene-start site in infected cells as there was no major contribution input polymerase to yield the mRNAs. Viral protein synthesis is of the leader gene or its varied U composition to the UV sensitivity required for replication to supply a source of N protein for of the I-60 mRNA in cells (Fig. 4). Consequently, these results have encapsidation of the nascent strand. These observations led to a considerable implications for the mechanism of regulation of gene model proposing RdRP activity was switched during Leϩ synthesis expression in VSV and, by extrapolation, other NNS RNA viruses. from transcription to replication by the availability of N protein (34, In addition, these experiments extend the utility of UV-mapping 35). While a requirement for N protein to encapsidate the nascent studies by demonstrating that the sequence of a genome can be RNA strand is indisputable (41), there is little evidence that N specifically altered to affect the target sensitivity of a downstream protein switches RdRP activity from transcription to replication. transcript. The finding that transcription and replication initiate at separate positions on the genome is incompatible with the possibility that N Transcription. The stop-start model of sequential transcription is switches RdRP activity from transcription to replication during widely accepted. This model states that the RdRP enters the synthesis of Leϩ. Rather, it suggests that transcription and repli-

9182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.152155599 Whelan and Wertz Downloaded by guest on September 28, 2021 cation are regulated through the use of separate initiation sites. findings show that the mutations engineered into the genome did Initiation site choice could be regulated by a modification to the not alter the mechanism of template recognition by polymerase RdRP, the template, or both. Polymerase initiating at the 3Ј end differentially between the viruses. Rather, they show that polymer- would synthesize leader RNA or the antigenome, a process that ase initiated at a separate site in vitro compared with in vivo. requires N protein for encapsidation (41, 42). The suggestion of two Precisely why transcription initiates at different positions in vivo and forms of polymerase is attractive, as specific mutations in polymer- in vitro was not determined, but possible explanations include the ase can separately affect transcription and replication (43–45). host-cell environment, the low-pH transition that occurs during The N RNA template could also regulate the site of polymerase infection of cells, and the abundance of M protein. Several host-cell initiation. The polR1 mutant of VSV has a single amino acid change factors may be required for RNA synthesis (47–50), and their in N (R146H), which perturbs the ratio of products that initiate at abundance likely differs in infected cells versus in vitro. The the 3Ј end or at the N gene-start in vitro (17). This change may alter pH-dependent transition during internalization of virions may alter the template structure, the ability of polymerase to bind to tem- RdRP template interaction. Altering the pH of in vitro transcription plate, or both. Cryoelectron microscopy identified four forms of the reactions alters the abundance of products that initiate at the 3Ј end Sendai virus template in infected cells (46). The form of the of the genome and read through the leader–N gene junction (17). template may influence the RdRP entry site, and polR1 may adopt Finally, the role of M in transcription is poorly understood; how- a conformation that favors internal entry. ever, it affects the ratio of RNA oligomers that correspond to Leϩ Previously we showed that the ability of a VSV subgenomic and N mRNA (51), and transcriptional attenuation (52). Deter- replicon to act as template for transcription or replication was mining which (if any) of these contribute to the difference in vitro influenced by the extent of complementarity between the genomic versus infected cells may reveal how RdRP initiation site choice is termini. Increased complementarity favored replication at the regulated. expense of transcription (31, 33). In view of the distinct entry sites In conclusion, we provided evidence that in infected cells, the for transcription and replication, it seems possible that polymerase polymerase of VSV initiates transcription directly at the first entry is influenced by the interaction of the termini, such that when gene-start sequence, and not through the prior transcription of a the termini interact the RdRP initiates replication at the extreme leader RNA. These experiments thus define separate initiation Ј 3 end of the genome, and when the termini do not interact, the points for transcription and replication on the VSV genome, and RdRP initiates transcription at the first gene-start site. thereby suggest a previously unrecognized means of regulation of the two processes. Transcription in Infected Cells Versus in Vitro. Previous work dem- BIOCHEMISTRY Ј onstrated that transcription initiated at the genomic 3 end in vitro We thank Andy Ball for unwavering enthusiasm throughout the project, (15), and we confirmed this for each recombinant virus in vitro M. Carpenter for statistical analysis, and J. Barr, S. Harmon, and E. (Figs. 2 and 3). However, in vivo, transcription was shown to initiate Hinzman for review. This work was supported by Grant R37-AI2464 internally at the first gene-start for each virus (Fig. 4). These from the National Institutes of Health to G.W.W.

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Whelan and Wertz PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9183 Downloaded by guest on September 28, 2021