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Copyright © 1997, American Society for Microbiology

A Novel Viral RNA Species in Sindbis Virus-Infected Cells

MATTHEW M. WIELGOSZ

AND

HENRY V. HUANG*

Department of Molecular Microbiology, Washington University School

of Medicine, St. Louis, Missouri 63110-1093

Received 3 March 1997/Accepted 8 August 1997

Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting

sequences and proteins involved in RNA transcription and replication. However, very little is known about how

these processes are coordinated. For example, synthesis of the genomic RNA and the subgenomic mRNA

depends on the minus strand. Do these activities occur independently on different templates, or can replication

and transcription take place simultaneously on the same template? We describe the appearance of a

SIN-specific, plus-sense RNA that is intermediate in size between the genomic and subgenomic RNA species. This

RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The

number of RNA II species, their sizes, and their abundances are influenced by the subgenomic promoter. We

have mapped the 3

*

end of RNA II to a site within the subgenomic promoter, four nucleotides before the

initiation site of the subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with

subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA

II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and

is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.

The alphaviruses belong to the Togaviridae family. They

have a global distribution and infect insect, avian, and

mam-malian hosts with various degrees of virulence (20, 46, 50, 62).

The identification of viral cis-acting sequences and trans-acting

proteins required for alphavirus replication and transcription

has led to the development of wide-host-range expression

vec-tors (4, 42, 73) and potential vaccines for disease-transmitting

mosquitoes (6).

In Sindbis virus (SIN), the first two-thirds of the incoming

genomic RNA is translated to yield two nonstructural

polypro-teins (nsPs), nsP123 and nsP1234. The polypropolypro-teins and/or

their proteolytic products constitute the viral contribution to

the RNA-dependent RNA polymerase (R-dRp). The exact

composition of the R-dRp is not known, and it may differ

depending on whether the R-dRp is engaged in replication or

transcription. However, several functions have been identified

or assigned to the individual nsPs which participate in viral

RNA synthesis. nsP1 is thought to be the enzyme that caps

viral RNA, since it can bind guanylate nucleotides (1) and

exhibits methyltransferase activity (45). nsP1 also functions in

minus-strand RNA synthesis (69). nsP2 has sequence motifs

characteristic of nucleoside triphosphate binding domains (17,

19, 27) and helicase domains (18). Indeed, nsP2 of Semliki

Forest virus (SFV) exhibits GTPase and ATPase activities

(52). However, attempts to demonstrate helicase activity in this

protein have not been successful (52). nsP2 also functions as

the nsP protease (68) and is critical to subgenomic mRNA

synthesis (24, 58, 59). Specific roles for nsP3 have not been

defined. nsP3 is serine/threonine phosphorylated in vivo (41)

and appears to be involved in genomic replication (24, 33, 35,

36, 64) and subgenomic mRNA transcription (33). nsP4

con-tains the GDD motif that is common to many viral RNA

polymerases (28) and is considered to be the polymerase of

SIN. Several temperature-sensitive (ts) mutations which affect

viral RNA synthesis have been mapped to nsP4 (23, 56), and

one of these renders the virus completely defective in RNA

synthesis (3, 30, 60).

The nsPs and/or their proteolytic products are believed to

assemble on the 3

9

end of the genomic RNA to synthesize a

complementary minus-strand RNA (31, 38). The minus strand

contains two cis-acting sequences that are critical to viral

rep-lication. The first is located at the 3

9

end of the minus strand,

and it directs the replication of genomic RNA, which, in turn,

is used for continued minus-strand synthesis and nsP

transla-tion (31, 40). The second cis-acting sequence is the subgenomic

promoter. It is located approximately 7.6 kb downstream from

the 3

9

end of the minus strand, and it is required for the

transcription of subgenomic mRNA (39, 47). Transcription

initiates at the promoter and by runoff transcription produces

a subgenomic mRNA that is 3

9

coterminal with the genomic

RNA. The subgenomic mRNA encodes the viral structural

proteins, and these package the genomic RNA into progeny

virions (22, 44, 67, 70, 71). As the SIN infection cycle

progresses, genomic RNA is routed predominantly into the

virion assembly pathway, concurrent with an increased rate of

plus-strand RNA synthesis, cessation of minus-strand

synthe-sis, and a decreased rate of nsP translation. The switch from

minus-strand synthesis early in infection to plus-strand

synthe-sis later in infection appears to involve the processing of the

nsPs into their individual nonstructural proteins and/or

pro-teolytic intermediates (12, 34–36).

It is not known how transcription and replication are

coor-dinated on the minus strand. There is some evidence that these

activities occur simultaneously on the same template (57, 63).

Three double-stranded (ds) replicative forms (RFs) have been

identified upon the treatment of partially ds replicative

inter-mediates with RNase (5, 13, 57, 63, 66). RF I constitutes a ds

form of the full-length genome, while RFs II and III

corre-spond to the ds forms of the first two-thirds (the nonstructural

region) and the remaining one-third (the structural region) of

the genome, respectively (66). Sawicki et al. found that an SFV

ts mutant exhibited decreased levels of subgenomic RNA

rel-ative to genomic RNA at the nonpermissive temperature and

displayed a correlated decrease in RFs II and III relative to RF

* Corresponding author. Mailing address: Department of Molecular

Microbiology, Washington University School of Medicine, Box 8230,

660 S. Euclid Ave., St. Louis, MO 63110-1093. Phone: (314) 362-2755.

Fax: (314) 362-1232.

9108

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I (57). Those authors concluded that a virus-specific protein

which functions to promote transcription and block genomic

RNA replication was responsible for the appearance of

RNase-sensitive sites within replicative intermediates, such that RNase

treatment of these species gave rise to RFs II and III (57).

Thus, when the virus-specific protein could not function

nor-mally at the nonpermissive temperature, the RNase-sensitive

sites of the replicative intermediates were masked and RF I

became the dominant species. Similar results were obtained by

Segal and Sreevalsan by using a ts mutant that was defective in

subgenomic mRNA transcription in SIN (63).

Although ds RNA can be obtained from infected cells upon

treatment with RNase, the predominant SIN-specific RNA

species in an infected cell are the single-stranded genomic and

subgenomic RNAs (65). Other single-stranded viral RNA

spe-cies have been observed in SIN (9, 26, 49, 53) and SFV (8, 29,

37). One species, in particular, was found to have a

sedimen-tation coefficient of 38S and an electrophoretic mobility

be-tween those of genomic and subgenomic RNAs under

nonde-naturing conditions (9, 37). When the SFV 38S species was

resolved under denaturing conditions, it was found to

comi-grate with the genomic 42S RNA (29). RNase T1

oligonucle-otide fingerprinting confirmed that the 38S species was most

likely a conformer of the genomic 42S RNA (29).

Recent observations in our laboratory (26) and others (49)

have shown that a glyoxal-denatured RNA species that is

in-termediate in size between genomic and subgenomic RNAs is

present in SIN-infected cells. This RNA, which we call RNA II,

may represent the single-stranded equivalent of RF II. Very

little is known about RNA II, other than that it is SIN specific

(unpublished observations) (49) and its appearance depends

on the SIN subgenomic promoter (26). We report here the

characterization of RNA II.

MATERIALS AND METHODS

Reagents, chemicals, isotopes, recombinant DNA materials, and methods.

Trizol reagent was obtained from Bethesda Research Laboratories (Grand Is-land, N.Y.). Cycloheximide was purchased from Sigma. [a-32P]GTP was from

Amersham (Arlington Heights, Ill.). [32P]orthophosphate and [g-32P]ATP were

obtained from ICN (Costa Mesa, Calif.). Restriction endonucleases and other recombinant DNA materials were purchased from New England Biolabs (Bev-erly, Mass.), Bethesda Research Laboratories, Epicentre Technologies (Madi-son, Wis.), or Boehringer Mannheim. Poly(A) polymerase was acquired from United States Biochemical (Cleveland, Ohio). A version of theDTaq DNA-dependent DNA polymerase was obtained at Washington University (St. Louis, Mo.), but the sequencing reaction was performed as described in theDTaq sequencing kit (United States Biochemical) for kinased primers. All molecular biology procedures were performed as described by Sambrook et al. (54), unless otherwise noted.

Cell lines.Unless otherwise noted, all vertebrate and invertebrate cells were grown at 37 and 30°C, respectively. Baby hamster kidney (BHK-21) cells (ATCC CCL10) were grown in minimal essential medium with Earle’s salts (MEM) supplemented with 10% heat-inactivated (HI) fetal calf serum (FCS). Cells between passages 5 and 15 were used. Primary chicken embryo fibroblasts (CEF) were prepared as described previously (48) and grown in MEM supplemented with 3% HI FCS, 100-U/ml penicillin, and 100-mg/ml streptomycin. Secondary CEF cells were used. C7-10 cells and C6-36 cells (ATCC 1660-CRL), originally isolated from Aedes albopictus larvae (55), were grown in MEM supplemented with 10% HI FCS and 10% tryptone phosphate buffer. Cells between passages 30 and 40 (C7-10) or 124 and 132 (C6-36) were used. African green monkey kidney (Vero) cells (ATCC 81-CCL) were grown in Medium 199 supplemented with 5% FCS (not HI). Cells between passages 125 and 132 were used.

Virus constructs.Toto1000 contains the entire SIN genome [11,703 nucleo-tides, excluding the poly(A) tail] downstream of the SP6 promoter (51). To-to1000 has one subgenomic promoter that is positioned;7.6 kb downstream from the virus’s 59 end. This promoter is required for transcription of the subgenomic mRNA which encodes the virion’s structural proteins. It is;4.2 kb in size and is 39coterminal with the genomic RNA.

TCS is similar to Toto1000, in that it contains the nonstructural (nsP) and structural (STR) coding regions of SIN. However, the nsP and STR coding regions of TCS are separated by the chloramphenicol acetyltransferase (CAT) gene (25). Additionally, TCS contains two subgenomic promoters (25). The first is the CAT promoter, and it is located;7.6 kb downstream from the 59end of

the genomic RNA (;12.6 kb). It is required for transcription of the CAT subgenomic mRNA, which is;5.0 kb in size and 39coterminal with the genomic plus-strand RNA. The second promoter is the STR promoter, and it is located

;8.4 kb downstream from the 59end of the genomic RNA. It is required for transcription of the STR subgenomic mRNA (identical to that of Toto1000), which is;4.2 kb in size and 39coterminal with the genomic plus-strand RNA. The CAT and STR promoters of TCS contain the298/114 subgenomic pro-moter sequences (298 refers to the number of nucleotides upstream of the start site of the subgenomic mRNA, and114 refers to the number of nucleotides downstream of the start site of the subgenomic mRNA).

The B2, III, and219/15 clones have been described previously and differ from TCS only in that their STR promoters are mutant (25, 26). The B2 STR pro-moter sequence was isolated from a library of viruses which contained random sequences between the213 and29 positions of the STR subgenomic promoter. The B2 virus has nucleotide changes at the213,210, and29 positions, where the wild-type A, G, and U nucleotides are replaced with G, A, and A, respec-tively. The III virus was another virus from the213 to29 library, and its STR promoter contained nucleotide changes at positions211 and210, where the wild-type G and G nucleotides have been replaced with C and C, respectively. The219/15 clone was constructed such that its STR subgenomic promoter contains the219 to15 promoter sequences in place of the298/114 promoter. TCIS is identical to TCS, except that a 979-bp insert is positioned immediately downstream of the CAT gene and immediately upstream of the STR promoter. Thus, the CAT subgenomic mRNA of TCIS is;6 kb in size and is 39coterminal with the genomic RNA. The STR subgenomic mRNA of TCIS is identical to that of Toto1000 (;4.2 kb in size and coterminal with the 39end of the genomic plus-strand RNA). TCIS was constructed as follows. TCS was digested with XhoI, and its ends were rendered blunt with T4 DNA polymerase and dephosphory-lated with calf alkaline phosphatase. A 979-bp insert was generated by digesting pSPORT1 (Bethesda Research Laboratories) with TaqI. The 979-bp DNA frag-ment was isolated, and its ends were rendered blunt. The XhoI-digested TCS vector and the 979-bp insert were ligated together by using T4 DNA ligase.

The240/114,240/15, and298/15 clones are identical to TCS, but their STR promoters (240/114,240/15, or298/15) have replaced the298/114 STR promoter of TCS. The construction of these clones was similar to that described for the219/15 clone (25), with some differences. First, the region of interest (i.e.,240/114,240/15, or298/15) was PCR amplified from the To-to1000 template by using oligonucleotides that contained the appropriate 59or 39

promoter sequences, flanked by either the XhoI or XbaI site, respectively. The PCR products were digested with XhoI and XbaI and directionally cloned into the Pneo S shuttle vector (25). The subcloned promoters were then digested with XhoI and BssHII and directionally cloned into TDV (25).

The219/114 clone differs from the219/15 clone in that the STR promoter contains the219/114 promoter in place of the219/15 promoter. The construc-tion of219/114 was done as follows. The219/15 clone was used as template DNA in the PCR amplification of a 211-nucleotide sequence using oligonucle-otides 1940 and 1914. Oligonucleotide 1940 (59CCCGTTTTCACCATGGGCA AATA 39) hybridizes to the noncoding strand of CAT 266 nucleotides upstream of the STR subgenomic mRNA start site. Oligonucleotide 1914 (59CTAGTCT AGAACTATGCTGACTATTTAGG 39) contains the25 to114 sequence and hybridizes to the coding strand of219/15, encompassing nucleotides25 to15 relative to the STR subgenomic RNA start site. The PCR product was digested with XhoI and XbaI and directionally cloned into the219/15 clone.

For the construction of TCS-21, Toto:ts 21 A2 was digested with BglII and ClaI and the 423-bp fragment was directionally cloned into TCS. Toto:ts 21 A2 contains a single G-to-A mutation at nucleotide 2590, changing Cys 304 of nsP2 to Tyr (24).

RNA transcription and transfection and preparation of viral stocks. SstI-linearized plasmids were transcribed in vitro with SP6 DNA-dependent RNA polymerase. The DNA templates were digested with DNase I, and the RNA transcripts were phenol, chloroform, and ether extracted prior to ethanol pre-cipitation. The transfection procedure was similar to that described by Liljestro¨m et al. (43). A 5-mg sample of a given transcript was mixed with 107BHK-21 cells,

and the cells were electroporated in a 0.2-cm gap electrocuvette using a T820 Electro Square Porator (BTX Inc., San Diego, Calif.). Transfected BHK-21 cells were added to 5 ml of growth medium, transferred to a culture dish, and incubated at 37°C for 16 h. The medium was collected and centrifuged at 12,0003g for 20 min at 4°C to pellet cell debris. Aliquots of the medium were placed into 0.5-ml microcentrifuge tubes and stored at280°C until use.

Infection and RNA labeling.Cells (;0.43106to 0.63106) were seeded onto

60-mm-diameter culture dishes. Vertebrate and invertebrate cells were main-tained at 37 or 30°C, respectively, for approximately 24 h. After this time, vertebrate cells approached 90% confluence while invertebrate cells approached 50% confluence. Vertebrate and invertebrate cells were both infected with virus at a multiplicity of infection (MOI) of 5 at either 37°C (vertebrate cells) or 30°C (invertebrate cells) in solution A (phosphate-buffered saline minus Mg21and Ca21) supplemented with 1% HI FCS. At 1 h postinfection (hpi), the inoculum was removed and the cells were washed twice with solution A (23°C) supple-mented with 1% HI FCS. The cell cultures then received 3 ml of the appropriate medium and were maintained at 37 or 30°C.

For RNA labeling in vertebrate cells, dactinomycin (1-mg/ml final concentra-tion) was added to the medium 1 h before labeling. The medium was then

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removed and replaced with 1.5 ml of labeling medium (1-mg/ml dactinomycin and 100-mC/ml [32P]orthophosphate in the appropriate phosphate-free cell

me-dium). The cells were incubated at 37°C for 2 h. RNA labeling for invertebrate cells was essentially the same as that for the vertebrate cells. Dactinomycin (5-mg/ml final concentration) was added to the medium 0.5 h before labeling (either 20.5 or 44.5 hpi). The medium was then removed and replaced with 1.5 ml of labeling medium (identical to the vertebrate labeling medium except for 5-mg/ml dactinomycin). The cells were incubated at 30°C for 3 h.

RNA isolation and analysis.The medium overlying each cell monolayer was removed, and the cells were washed twice with ice-cold solution A. The cells were solubilized with 3 ml of Trizol reagent and stored at280°C. RNA was isolated in accordance with the manufacturer’s protocol and dissolved in 10 to 30ml of diethylpyrocarbonate-treated water. Approximately 1/10 of the total RNA from a given cell sample was denatured with glyoxal and dimethyl sulfoxide (7) and electrophoresed through 1% agarose gels in 10 mM sodium phosphate (pH 7.0) (54). Gels were fixed with methanol and dried.32P-labeled RNA was detected by

autoradiography with film that was flash hypersensitized up to an A440of;0.15

above that of nonflashed film (32, 54). Exposures were done at280°C for 1 to 5 days without an intensifying screen.

32P-labeled RNA was quantitated on a Bio-Rad PhosphorImager. The

back-ground counts were estimated by the average counts above and below those of the RNA species and subtracted from the counts for the RNA species. The relative molar quantity of each viral RNA was determined by multiplying its background-subtracted counts by a size correction factor. For example, the relative amount of the structural subgenomic RNA of TCS compared to genomic RNA was calculated by multiplying the actual counts of the subgenomic RNA by 3.03 (length of TCS genome divided by the length of the STR subgenomic RNA, i.e., 12,589/4,156).

Northern blot analysis.The Northern blotting procedure was performed as previously described (54), with minor changes. Samples (3mg) of total RNAs from TCS-, Toto1000-, and mock-infected cells were denatured and resolved on a 1% agarose gel. The gel was soaked in 20 mM NaOH for 20 min and then washed extensively with water. The gel was neutralized with 203SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and the RNA was blot transferred overnight to a Hybond N1membrane (Amersham). The membrane was dried at room temperature and irradiated with 120 mJ of UV light (254 nm) with a Stratagene auto-cross-linker (Stratagene, La Jolla, Calif.). All probes used in the Northern analyses were in vitro transcribed from DNA templates by using the T7 DNA-dependent RNA polymerase and were radiolabeled with [a-32P]GTP. The

STR probe template contains the capsid coding sequences of Toto1000 between nucleotides 7612 and 7871 cloned into the HindIII site of pSPORT1. The CAT probe template contains nucleotides 195 to 442 of the bacterial CAT gene (2) directionally cloned into pSPORT1 at the BamHI and EcoRI sites. The non-structural (NSP) probe template contains the nsP4 coding sequences of To-to1000 between nucleotides 7338 and 7593 cloned at the HindIII site of pSPORT1. The runoff site for the STR and NSP probes was MluI, and that for the CAT probe was BamHI. The RNA probes were each of minus-sense polarity. To reprobe a blot, probes were removed from the membrane by twice pouring an

;98°C solution of 1% sodium dodecyl sulfate onto the membrane.

Poly(A) tailing.Total RNA (4mg in a volume of 7ml) was incubated at 50°C for 10 min, and then cooled on ice before the addition of 8ml of poly(A) reaction buffer [37.5 mM Tris HCl [pH 7.0], 1.68 mM MnCl2, 3.0 mM MgCl2, 1.87 mM

ATP, 94mM dithiothreitol, 1.5-mg/ml bovine serum albumin, 112.3 mM KCl, 18.7% glycerol, 18.8-U/ml poly(A) polymerase]. Poly(A) reactions were carried out at 30°C for 20 min with 10-U/ml (final concentration) poly(A) polymerase and were stopped by addition of phenol. Total RNA was chloroform and ether extracted and then ethanol precipitated with a tRNA carrier.

RT and PCR.Total RNA from the poly(A) reactions was solubilized in 10ml of a NotI oligo(dT) mixture [32mM NotI oligo(dT); 59GATCTAGAGCGGCC GCCCTTTTTTTTTTTTTTT 39in diethylpyrocarbonate-treated water), heated at 70°C for 10 min, and then cooled on ice. The reverse transcription (RT) reactions were performed in accordance with the Superscript II protocol at 48°C for 1 h. The reactions were terminated by heat denaturation at 70°C for 10 min. Half of each RT reaction mixture was used in a 100-ml PCR mixture with the 1940 and NotI oligo(dT) oligonucleotides at 1mM as primers and 2.5 U of Tth polymerase. Thirty-five PCR cycles were performed (one cycle was 40 s at 95°C, 30 s at 60°C, and 40 s at 72°C).

Cycle sequencing analysis.PCR-amplified products were resolved on 2.5% NuSieve GTG gels and gel purified (54). A 5-fmol sample of the PCR product was used per dideoxynucleotide termination reaction, in accordance with the

DTaq cycle sequencing kit instructions for kinased primers. The219/15 PCR product was sequenced with the 1941 primer (59GAATTACAACAGTACTGC GATGA 39), which hybridizes to the noncoding strand of CAT 152 nucleotides upstream of the STR subgenomic mRNA start site. The PCR products obtained for the XbaI TCS transcript, TCS, and B2 samples were sequenced with primer 1913 (59 CCGCTCGAGGGGGCCCATTACACCTGTCCTAC 39), which hy-bridizes to the noncoding strand of TCS, encompassing nucleotides298 to283 relative to the STR subgenomic mRNA start site. The sequencing reaction products were resolved on 8% denaturing polyacrylamide (19:1 acrylamide-bisacrylamide ratio) gels (54) and analyzed by autoradiography.

RESULTS

Gel analyses of SIN intracellular RNA reveal two prominent

bands: the 49S genomic and 26S subgenomic RNAs. A third

RNA species is frequently observed (see Toto1000 in Fig. 1A).

We call this RNA II. Similar RNAs have been observed and

shown to be isomers of the 49S genomic RNA (e.g., through

denaturation) (29). We previously showed that insertion of an

additional promoter into the SIN genome results in the

tran-scription of an additional mRNA. The new mRNA, like the

normal SIN subgenomic mRNA, initiates at the promoter and,

by runoff transcription, produces a new subgenomic mRNA

that is 3

9

coterminal with the genomic RNA (see CAT and

STR subgenomic mRNAs in Fig. 1A). Interestingly, these

vi-ruses produce two species of RNA II. Indeed, several physical

characteristics of RNA II are determined by the subgenomic

promoter (26). Toto1000 has one subgenomic promoter which

directs the transcription of the STR subgenomic RNA. TCS

and its derivatives (B2,

2

19/

1

5, III, and TCIS) have two—the

CAT promoter, which directs the transcription of the CAT

subgenomic RNA, and the STR promoter, which directs the

transcription of the STR subgenomic RNA. As shown in Fig.

1A, Toto1000 has only one RNA II species, while TCS and its

derivatives have two. These results show that the number of

different RNA II species in the cell correlates with the number

of promoters in the infecting virus.

The second property of RNA II that is affected by the

sub-genomic promoter is size. The STR promoter of Toto1000 and

the CAT promoters of TCS and its derivatives are each

[image:3.612.309.547.71.306.2]

posi-tioned

;

7.6 kb downstream from the 5

9

ends of each virus.

FIG. 1. Properties of RNA II that are determined by the subgenomic pro-moter. (A) BHK-21 cells were infected with the appropriate virus at an MOI of 5 and treated with dactinomycin 1 h before labeling with [32P]orthophosphate at

3 hpi. At 5 hpi, total RNA was isolated, denatured, and resolved on a 1% agarose gel. Exposures were at280°C for 2 to 5 days. G is genomic RNA. Lg and Sm are the large and small RNA II species, respectively. The CAT and STR subgenomic RNAs are designated CAT and STR, respectively. (B) Same as in A but the in vivo-incorporated label of each RNA (relative to the genomic RNA of the virus) was quantitated on a Bio-Rad PhosphorImager and used to calculate the STR/ CAT and Lg/Sm ratios shown. These relative molar ratios have been normalized

against those obtained for TCS.

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Accordingly, the sole RNA II species of Toto1000 and the

small RNA II species of TCS and its derivatives have identical

mobilities, corresponding to a size of 7.6 kb (Fig. 1A). The

same relationship holds for the STR promoters of TCS and its

derivatives, which are positioned

;

8.43 kb downstream from

each virus’s 5

9

end (except for that of TCIS). The STR

pro-moter of TCIS is positioned 979 nucleotides downstream from

the original STR promoter position of TCS. As shown in Fig.

1A, the new position of the STR promoter correlated with an

increase in the size of the large RNA II species, from

;

8.43 to

;

9.41 kb. These results not only demonstrate that the size of

RNA II is determined by the position of the subgenomic

pro-moter but also suggest that RNA II is colinear with the 5

9

end

of genomic RNA and has a 3

9

end that is close to the position

of the subgenomic promoter.

A third characteristic of RNA II that is affected by the

subgenomic promoter is abundance. We previously found that

the abundance of RNA II decreases when the strength of the

subgenomic promoter decreases (26). Indeed, the

2

19/

1

5

vi-rus, whose STR promoter contains the

2

19/

1

5 promoter

se-quence in place of the

2

98/

1

14 STR promoter of TCS, has a

weaker STR promoter than TCS (as assessed by the relative

decrease in the STR-to-CAT subgenomic RNA ratios), and

this correlated with decreased large (Lg)-to-small (Sm) RNA

II ratios compared to TCS (Fig. 1A). Similar results were

obtained with the B2 virus, which contains a weaker STR

promoter than TCS (see Materials and Methods). The relative

molar ratio of STR to CAT subgenomic mRNAs for B2 is

;

0.6

(as assessed by PhosphorImager scans), while that for TCS is

1.9. Correspondingly, the ratio of Lg to Sm RNA II for B2 is

;

0.5, while that for TCS is

;

1.5. In contrast, the STR

pro-moter of virus III has activity comparable to that of the TCS

STR promoter (26), where the relative molar ratios of STR to

CAT subgenomic mRNAs are 1.7 for III and 1.9 for TCS. This

corresponded to a Lg-to-Sm RNA II ratio similar to that

ob-tained for TCS (

;

1.0 for III and

;

1.5 for TCS).

We explored the association of RNA II abundance and

subgenomic promoter activity further, with derivatives of TCS

that contained various deletions in their respective STR

pro-moters. The

2

40/

1

14,

2

40/

1

5,

2

98/

1

5, and

2

19/

1

14 viruses

are identical to TCS, except that their STR promoter

se-quences have replaced the

2

98/

1

14 STR promoter of TCS. As

shown in Fig. 1B, the Lg-to-Sm RNA II ratios of the

2

19/

1

5

and

2

19/

1

14 viruses are lower than that obtained for TCS,

and these correlate with decreased STR-to-CAT subgenomic

mRNA ratios (relative to TCS). Note that the STR/CAT and

Lg/Sm ratios listed in Fig. 1B are normalized against those

obtained for TCS. The addition of the

1

6 to

1

14 promoter

sequences to the minimal

2

19/

1

5 promoter in

2

19/

1

14

de-creased its STR-to-CAT subgenomic mRNA ratio nearly

threefold compared to that of

2

19/

1

5, and this corresponded

to a Lg-to-Sm RNA II ratio that was lower than that obtained

for

2

19/

1

5. In contrast, the STR-to-CAT subgenomic mRNA

ratios of the

2

40/

1

5 and

2

98/

1

5 viruses are not appreciably

different from that of

2

19/

1

5, but their Lg-to-Sm RNA II

ratios are four- to fivefold greater than that of

2

19/

1

5 (Fig.

1B). These results indicate that the

2

40 to

2

20 promoter

sequences, when placed upstream of the minimal

2

19/

1

5

pro-moter, can increase the abundance of RNA II in a way that

does not appear to be related to promoter strength.

Interest-ingly, the

2

40 to

2

20 promoter sequences do enhance

pro-moter strength when they are placed upstream of the

2

19/

1

14

STR promoter. Indeed, the activity of the

2

40/

1

14 STR

pro-moter is nearly indistinguishable from that of the

2

98/

1

14

STR promoter of TCS (Fig. 1B).

Appearance of RNA II in different cells.

RNA II has

previ-ously been observed in BHK-21 hamster and C7-10 mosquito

cells (25, 26). We used Vero, CEF, and C6-36 cells as

addi-tional hosts for infection by TCS and analyzed the appearance

of RNA II at different times during the infection cycle. The

times at which total RNA was harvested were determined by

selecting the stages of TCS growth that were comparable

among the different cell types. As shown in Fig. 2, RNA II was

detected in all of the vertebrate (Fig. 2A) and invertebrate

(Fig. 2B) cells tested, both early and late in infection. The

mock-infected samples of the vertebrate and invertebrate cells

did not yield any radiolabeled RNA species similar in size to

RNA II (data not shown). RNA II was observed in BHK-21

cells infected at 30°C and as early as 3 or 8 hpi in BHK-21 and

C7-10 cells, respectively (data not shown). These experiments

demonstrate that the appearance of RNA II is not limited to a

particular cell type, stage of infection, or temperature and

suggest that the appearance of this RNA in infected cells is a

general phenomenon. We performed several experiments like

those depicted in Fig. 2 to measure the relative molar

abun-dance of RNA II compared to the genomic and subgenomic

RNAs of TCS and Toto1000. Intracellular viral RNAs were

labeled for 2 h before isolation and gel analysis. The results are

summarized in Table 1. Under these conditions, the relative

molar abundance of RNA II changes by about twofold

be-tween 5 and 8 hpi (Table 1). On average, there is

approxi-mately 1 RNA II species to every 7 genomic or 50 subgenomic

RNAs in Toto1000-infected cells. These are minimal

esti-mates, as the stability of RNA II may be less than that of the

other viral RNAs.

Northern blot analysis of RNA II.

We previously found that

a negative-sense probe complementary to the 5

9

end of

[image:4.612.321.534.70.307.2]

genomic RNA could detect RNA II by Northern blot

hybrid-ization (unpublished results). This indicated that RNA II is of

FIG. 2. Appearance of RNA II in different vertebrate (A) and invertebrate (B) cells, both early and late in infection. Cells were infected with TCS at an MOI of 5 and treated with dactinomycin for 1 h (vertebrate) or 0.5 h (invertebrate) before labeling with [32P]orthophosphate. The cells were labeled for 2 h

(verte-brate) or 3.5 h (inverte(verte-brate) before total RNA was isolated at the times shown. For definitions of abbreviations, see the legend to Fig. 1.

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plus-sense polarity and suggested that its 5

9

end is coterminal

with genomic RNA. Furthermore, the results of Fig. 1A

sug-gested that the 3

9

end of RNA II is close to the position of the

subgenomic promoter. We determined the boundary of each

RNA II species in TCS- and Toto1000-infected cells by

North-ern blot analysis using negative-sense probes that hybridize to

positions both upstream and downstream of the subgenomic

promoter.

We first used an STR probe that is complementary to

se-quences immediately downstream of the STR subgenomic

pro-moter, between nucleotides

1

15 and

1

259 of Toto1000 or

nucleotides

1

21 and

1

264 of TCS. There are 35 nucleotides in

the STR subgenomic untranslated region (downstream of the

STR promoter) which are identical to those in the CAT

sub-genomic untranslated region (downstream of the TCS CAT

promoter). The Lg RNA II species of TCS contains these

nucleotides and may be detected by the STR probe. As shown

in Fig. 3A, the STR probe detected the genomic RNA of each

virus, as well as the CAT subgenomic mRNA of TCS and the

STR subgenomic mRNA of both viruses. However, the STR

probe failed to detect the RNA II species of either virus. This

result indicates that the 35 nucleotides of complementarity

between the Lg RNA II species of TCS and the STR probe was

insufficient for its detection and suggests that any RNA II

species whose 3

9

end extends beyond the STR subgenomic

promoter to include only the 35 nucleotides of the STR or

[image:5.612.53.548.81.153.2]

FIG. 3. Northern blot analysis of RNA II. BHK-21 cells were infected with Toto1000 or TCS at an MOI of 5 or mock infected. Total RNA was harvested at 5 hpi. RNA (3mg per sample) was resolved on a 1% agarose gel and transferred to a nylon membrane. The membrane was probed with an in vitro-transcribed minus-sense STR probe (A), CAT probe RNA (B), or NSP probe (C). (D) Target RNAs of the different probes for TCS between nucleotides 7250 and 8750. The coding regions of TCS are designated NSP, CAT, and STR and are shown as filled boxes (black, textured gray, and white, respectively). The white boxes represent the untranslated region downstream of the CAT and STR promoters. The CAT and STR promoters are represented by arrows and the subscript P. The hash marks designate 150-nucleotide intervals. The open area upstream of the STR promoter represents the XhoI and ApaI sites of TCS. The probes are labeled and represented as shaded boxes which correspond to their target sequences. For definitions of abbreviations, see the legend to Fig. 1.

TABLE 1. Relative molar ratios of RNA II in TCS and Toto1000-infected BHK-21 cells at 37°C

Virus, hpi Ratio6SD

a

nb

G/STR G/CAT G/Sm G/Lg STR/Sm STR/Lg CAT/Sm

Toto1000, 5

0.10

6

0.03

5

6

1

47

6

10

3

Toto1000, 8

0.14

6

0.04

8

6

2

55

6

6

3

TCS, 5

0.33

6

0.05

0.56

6

0.14

15

6

4

18

6

4

59

6

10

27

6

8

5

TCS, 8

0.25

6

0.07

0.56

6

0.16

10

6

4

10

6

1

37

6

7

17

6

6

3

aThe in vivo incorporated label of each RNA species (relative to the genomic RNA of the virus) was quantitated on a Bio-Rad PhosphorImager and used to calculate

the relative molar ratios shown. G refers to the genomic RNA, STR and CAT refer to the STR and CAT subgenomic RNAs, and Sm and Lg refer to the small and large RNA II species, respectively. SD, standard deviation of each RNA ratio, from n experiments.

bn, number of independent experiments performed.

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CAT subgenomic untranslated region (which are

complemen-tary to the STR probe) would not be detected. Thus, the 3

9

end

of RNA II does not extend significantly beyond the STR

pro-moter.

We removed the STR probe from the membrane and

re-probed the blot a second time with a CAT probe that is

com-plementary to the 5

9

untranslated region of CAT and a portion

of the CAT coding region between nucleotides 7657 and 7876

of TCS, several hundred nucleotides upstream of the STR

promoter and 45 nucleotides downstream of the CAT

pro-moter. As shown in Fig. 3B, the genomic and CAT subgenomic

RNA species of TCS were detected but the STR subgenomic

mRNA of TCS was not. The CAT probe did not detect any

RNA species in the Toto1000 sample, since this virus does not

contain any CAT sequence. The CAT probe did detect the Lg

RNA II species of TCS but failed to detect the Sm RNA II

species. This indicates that the 3

9

end of the Lg RNA II species

extends sufficiently beyond the CAT promoter for its detection

but the 3

9

end of the Sm RNA II species does not.

To map the 3

9

end of RNA II more precisely, we stripped off

the CAT probe and reprobed the blot a third time with an NSP

probe that is complementary to nucleotides

2

263 to

2

5 of the

subgenomic promoter. The STR and CAT promoters of TCS

have identical sequences between nucleotides

2

98 and

2

5.

Thus, the NSP probe is complementary to 93 nucleotides of the

CAT subgenomic mRNA of TCS and may detect this species.

The results presented in Fig. 3C demonstrate that the genomes

of TCS and Toto1000 were detected, as expected. The NSP

probe did not hybridize to the STR subgenomic mRNA but did

give a signal for the CAT subgenomic mRNA of

TCS—indi-cating that the 93 nucleotides of complementary between the

NSP probe and the CAT subgenomic mRNA of TCS were

sufficient for its detection. The NSP probe also detected the

RNA II species of each virus. This result, in combination with

those obtained from Fig. 3A and B, demonstrates that the 3

9

end of each RNA II species lies in close proximity to the

position of the subgenomic promoter.

3

*

end of RNA II.

Because the abundance of RNA II is low

(Table 1), we polyadenylated the 3

9

end of this species to

facilitate its amplification for sequence analysis. A control

ex-periment was first performed by using runoff transcripts that

were synthesized from the XbaI-digested TCS plasmid. In

TCS, the XbaI site is 15 nucleotides downstream of the STR

promoter. Thus, transcripts which terminate at this site are

similar in size to the large RNA II species of TCS. We used 100

fmol of transcript in two separate polyadenylation reactions,

either in the presence or in the absence of RNA from

unin-fected BHK-21 or C7-10 cells. After the polyadenylation

reac-tions, the NotI oligo(dT) primer was used to prime cDNA

synthesis and the products were PCR amplified by using the

1940 and NotI oligo(dT) primers (Fig. 4A). Any RNA having

a free 3

9

-OH should be polyadenylated and competent for

subsequent cDNA synthesis. However, only those products

which contain CAT sequences complementary to the 1940

primer should be amplified. The distance from the 5

9

end of

the 1940 primer to the XbaI runoff site is 284 nucleotides, and

the length of the NotI oligo(dT) primer is 33 nucleotides.

Consequently, a PCR product of no less than 317 bp was

expected (Fig. 4A). Indeed, we obtained from each transcript

sample (either in the presence or in the absence of RNA from

uninfected cells) a PCR product that was slightly larger than

310 bp (data not shown). The results of Fig. 4B show that the

sequence of each PCR product is identical to that of the TCS

STR promoter and the poly(A) tail was added exactly at the

XbaI site.

We then treated RNA from TCS-infected cells with this

amplification method. Total RNA (4

m

g) was used in the

poly-adenylation reaction, and RT-PCR was performed as

de-scribed before. A PCR product of approximately 300 bp was

generated (Fig. 4A). The results presented in Fig. 4C show that

the sequence of the PCR product was that of the TCS STR

promoter, and the poly(A) tail was added to position

2

4 of the

STR promoter. In related experiments, we found that the small

RNA II species of TCS and the large RNA II species of

TCS-infected CEF and C7-10 cells have sequences identical to

that of the STR subgenomic promoter, with poly(A) tails

added to position

2

4 of the subgenomic promoter (data not

shown). Taken together, these results suggest that similar

mechanisms generate the 3

9

end of RNA II in different cell

types and different subgenomic promoters.

We also tested if the 3

9

end of the large RNA II species in

2

19/

1

5- and B2-infected cells were different from that

ob-tained with TCS. The size of the PCR product of the B2

sample was approximately 300 bp, while that of

2

19/

1

5 was

;

220 bp (since nucleotides

2

98 to

2

20 of the STR promoter

are deleted) (data not shown). The abundance of the B2 and

2

19/

1

5 PCR products was lower than that of the TCS product

(data not shown), probably because these viruses have

rela-tively less of the Lg RNA II species than the Sm RNA II

species, compared with TCS (Fig. 1A). As shown in Fig. 4D,

the sequence of each PCR product is identical to that of the

STR promoter of each virus. In both cases, the poly(A) tail was

added to position

2

4 of the STR promoter. These results

indicate that the

2

19/

1

5 subgenomic promoter alone, in the

absence of flanking promoter sequences, determines the 3

9

end

of RNA II. Similarly, nucleotide substitutions at positions

2

13,

2

10, and

2

9 of the B2 STR promoter do not affect the 3

9

end

of RNA II.

Possible origins of RNA II.

Since RNA II is of plus-sense

polarity (Fig. 3 and 4), it is conceivable that it might originate

from the cleavage of genomic RNA at the position of the

subgenomic promoter at any time during the isolation or

anal-ysis of viral RNA. We tested this possibility by adding

radio-labeled TCS transcripts to Trizol-solubilized monolayers of

infected or mock-infected cells. The results of Fig. 5A show

that the samples which received TCS transcripts did not

gen-erate prominent, RNA II-like species and indicate that the

procedures used to isolate and analyze the viral RNA of TCS

do not cleave genomic RNA at the position of the subgenomic

promoter to yield significant amounts of RNA II-like species.

It should be noted that RNA species similar in size to the STR

subgenomic mRNA of TCS do accumulate, and these species

appear to be in vitro transcripts that prematurely terminate

within the nsP coding region (as assessed by Northern blot

analysis).

The generation of RNA II by some unknown mechanism

may require active transcription at the subgenomic promoter,

since the appearance of RNA II is correlated with subgenomic

mRNA synthesis (Fig. 1 and 2). Thus, inhibition of subgenomic

mRNA synthesis should prevent the appearance of RNA II.

This hypothesis was tested two ways.

Cycloheximide prevents or limits the formation of first-time

subgenomic mRNA transcription complexes during the early

part of infection but does not affect replication complexes

which have formed prior to treatment (61). We used this drug

between 2 and 3 hpi (a time at which the formation of

sub-genomic transcription complexes should be sensitive to

cyclo-heximide treatment) to observe its effect on the appearance of

RNA II. At 5 hpi, total RNA was isolated. As shown in Fig. 5B,

the samples that received cycloheximide within the first 140

min pi had substantially less subgenomic RNA than did

sam-ples receiving later treatments. This correlated with a decrease

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in the abundance of RNA II to nearly undetectable levels. At

2 h 40 min pi, the effect of cycloheximide on subgenomic

mRNA synthesis was minimal and this corresponded to an

increased abundance of RNA II compared to that in samples

treated earlier. At 3 hpi, the effect of cycloheximide was not

apparent. Indeed, the relative abundances of the subgenomic

mRNAs and RNA II to the genomic RNA for the 3-h

cyclo-heximide sample was indistinguishable from that of the

un-treated sample. We also found that cycloheximide-un-treated

samples that were isolated at 3 versus 5 hpi exhibited similar

patterns of inhibited subgenomic mRNA synthesis, and this

correlated with a decrease in the abundance of RNA II (data

not shown). These results suggest that treatment with

cyclo-heximide during the early part of the infection cycle inhibits

the translation of proteins that are required for subgenomic

mRNA synthesis and correspondingly reduces the abundance

of RNA II to nearly undetectable levels. The results of Fig. 5B

also suggest that genomic RNA, despite having an increased

abundance relative to subgenomic mRNA in this experiment,

is not cleaved during the isolation and analysis of viral RNA to

yield RNA II.

[image:7.612.66.523.98.476.2]

The second method we employed to determine the effect of

defective subgenomic mRNA synthesis on the abundance of

RNA II involved the use of a mutant virus. Toto:ts 21 A2 is

RNA negative at the nonpermissive temperature (58) and is

defective in subgenomic mRNA synthesis in a

temperature-independent manner (11). The causal mutation of Toto:ts 21

A2 maps to residue 304 of nsP2 but does not affect nsP

pro-cessing (24). We introduced this mutation into the TCS

con-struct to create TCS-21. Cells were infected with TCS and

FIG. 4. Sequence determination of the 39end of RNA II. (A) Primers used to amplify and sequence the 39end of the XbaI TCS transcript and the Lg RNA II species of TCS. The 1940 and NotI oligo(dT) primers were used for RT-PCR amplification, and the 1913 primer was used for sequencing. The 59ends of each primer are shown as gray-filled circles. The lines that are labeled RNA or single-stranded DNA (ss DNA) represent intermediates used in the amplification procedure for each sample but are scaled with respect to the Lg RNA II species of TCS. The coding regions in each PCR product are represented by filled boxes (textured gray for CAT and black for NSP). The open box for the XbaI TCS transcript represents the region downstream of the STR subgenomic mRNA start site (indicated with an arrow). The cDNA species are extended minimally by 33 nucleotides [the length of the NotI oligo(dT) primer]. The line which makes up the right side of each PCR product represents this 33-nucleotide extension. (B) 39-end sequence of XbaI-linearized TCS transcripts, either in the presence or in the absence of RNA from C7-10 cells. Each sequence reads from left to right (G, A, T, and C, respectively). (C) 39-end sequence of the large RNA II species of TCS. (D) 39-end sequences of the large RNA II species of B2 and219/15, respectively.

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TCS-21 at either 30°C (in duplicate) or 40°C. Duplicate cell

cultures which were infected at 30°C were shifted to 40°C at 4

hpi. At 8 hpi, total RNA was isolated from each sample. As shown

in Fig. 5C, the TCS-21-infected samples maintained at 30°C or

shifted to 40°C were noticeably defective in subgenomic mRNA

synthesis compared to those of TCS. This corresponded to a

substantial decrease in the abundance of RNA II for TCS-21,

relative to that of TCS. The TCS-21 sample infected and

main-tained at 40°C exhibited defective RNA synthesis for all viral

RNAs. These results demonstrate that an alteration in the coding

region of nsP2 adversely affects subgenomic mRNA transcription,

and this correlates to a reduction in the abundance of RNA II.

These results are also consistent with those in Fig. 5B and further

support the hypothesis that genomic RNA is not cleaved at the

position of the promoter to generate RNA II during the isolation

and analysis of viral RNA.

DISCUSSION

The results presented here demonstrate that RNA II is of

plus-sense polarity and show that its 3

9

end maps to the

2

4

position of the subgenomic promoter. We do not know how the

promoter acts to give rise to RNA II. It is possible that RNA

secondary structure in the promoter causes the generation of

RNA II. However, the 3

9

end of RNA II is the same, whether

it terminates at the TCS, B2, or

2

19/

1

5 promoter. Thus, any

secondary structure which generates RNA II must be present

in the minimal promoter itself and be unaffected by mutations

in the B2 promoter. Computer analyses failed to detect any

secondary structure in the minimal promoter.

Alternatively, the promoter may act indirectly to facilitate

the binding of specific proteins which cause the generation of

RNA II. The observation that both cycloheximide treatment

early in infection and the mutation of TCS-21 reduce the

amount of subgenomic RNA synthesized relative to genomic

RNA, with a corresponding decrease in the abundance of

RNA II, implies that proteins are more likely to be involved in

the generation of RNA II than is secondary structure inherent

to the promoter.

There are several mechanisms by which proteins bound at

the subgenomic promoter might cause the generation of RNA

II. One class of models invokes RNA cleavage as a possible

mechanism. Proteins bound at the promoter might stimulate

the cleavage of any genomic RNA (at the

2

4 position of the

promoter) that becomes at least locally hybridized to the minus

strand. Another possibility is that proteins may recognize and

cleave the subgenomic promoter at position

2

4 or close to it

on the minus strand. Thus, the replication of genomic RNA on

cleaved minus-strand templates would then terminate at

posi-tion

2

4. Yet another mechanism is that proteins bound to the

promoter cleave nascent genomic RNA once a replication

complex reaches the promoter. All of the cleavage mechanisms

must explain the specificity of cleavage to leave a free 3

9

-OH at

position

2

4 of the promoter.

Although the generation of RNA II via a cleavage

mecha-nism cannot be ruled out, we favor a model in which the

generation of RNA II is a consequence of competition

be-tween the replication of genomic RNA and the initiation of

subgenomic mRNA synthesis. In this model, it is assumed that

replication and transcription occur simultaneously on the same

minus-strand template and that subgenomic transcriptional

initiation is slow compared to genomic RNA elongation. Three

outcomes can be envisioned. First, replication complexes may

physically displace the proteins bound to the promoter that

were engaged in transcriptional initiation. The net effect may

be a reduction in the levels of transcribed subgenomic RNA,

and RNA II would not be generated. Second, replication

com-plexes may pause at the promoter because initiation comcom-plexes

at the promoter would physically block continued genomic

RNA elongation. Once transcription had initiated, replication

complexes could then resume elongation. The net effect may

be a reduction in the levels of replicated genomic RNA. When

cells are lysed for RNA isolation, the partially completed

genomic RNAs in the stalled replication complexes are

re-leased as RNA II molecules. Finally, replication complexes

may pause on the promoter but then dissociate from the

mi-nus-strand template. Once dissociated, the replication

com-plexes may fall apart and release their nascent genomic RNA

as RNA II. RNA II molecules may therefore be dead-end

products. It is interesting that if the pausing or stalling model

is correct, then the 3

9

end of RNA II is essentially a “toe print”

[image:8.612.55.280.69.391.2]

of whatever protein was bound to the promoter. Given the

abundance of RNA II relative to the genomic RNA (one to

FIG. 5. Possible origins of RNA II. (A) Nonradioactively labeled TCS and mock-infected BHK-21 cells were solubilized with Trizol at 5 hpi. Full length in vitro transcripts of TCS (radiolabeled with [a-32P]GTP) were immediately added

to each sample, and RNA was analyzed as described in Materials and Methods). The TCS RNA sample (labeled in vivo with [32P]orthophosphate) was used as a

standard to determine the positions of the Lg and Sm RNA II species. (B) BHK-21 cells were infected with TCS and labeled as described in the legend to Fig. 1. At the times listed, cycloheximide was added to samples marked with a plus sign (final concentration, 10mg/ml). Total RNA was isolated at 5 hpi and analyzed as described in the legend to Fig. 1A. (C) TCS and TCS-21 were used to infect BHK-21 cells at the temperatures shown. At 4 hpi, duplicate cell cultures grown at 30°C were shifted to 40°C (labeled 30-40). All cell cultures were treated with dactinomycin 1 h before labeling with [32P]orthophosphate. Cell labeling was

terminated 2 h later, at 8 hpi, and total RNA was isolated and analyzed as described before. For definitions of abbreviations, see the legend to Fig. 1.

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every seven; Table 1), both pausing scenarios predict that the

promoter poses a significant impediment to genomic RNA

synthesis, and this may be a novel mechanism to coordinate

replication and transcription. Alternatively, the dissociated

replication complex may bind to another minus-strand

tem-plate to resume and complete the synthesis of the nascent

genomic RNA. In this case, RNA II may be a precursor to a

recombinant genome that is generated during plus-strand

syn-thesis.

Regardless of the mechanism of RNA II generation, RNA II

may have an effect on the SIN infection cycle. For example,

RNA II may or may not be capped or polyadenylated in vivo,

but there are examples in the literature in which RNA species

devoid of a 5

9

cap, poly(A) tail, or both are translation

com-petent (10, 15, 16, 21, 72). If RNA II were translated, nsP4

would be deficient in at least two amino acids at its carboxy

terminus. Modified nsP4 could function differently from

full-length nsP4 with respect to viral RNA synthesis. Some of these

differences may include altered cis-acting sequence specificity

or processivity. Another effect RNA II may have on the SIN

infection cycle is decreasing the number of virion particles with

full-length genomic RNA. The 3

9

end of RNA II extends well

beyond the putative encapsidation signal (70), so it is not

unreasonable to suggest that it can be packaged. Indeed, an

RNA species similar in size to that expected for RNA II has

been observed in virion particles (14). If RNA II is packaged

and is present in a molar ratio of 1 to 10 compared to genomic

RNA (Table 1), a 10% decrease in the number of virion

par-ticles that contain full-length genomic RNA could lower the

efficiency with which a virus population initiates infection.

Fi-nally, as mentioned before, RNA II may be a precursor to a

recombinant genome. Recombination is one of several

mech-anisms that a virus population can employ to increase its

di-versity. If template switching for a replication complex

oc-curred (due to a block imposed by initiation complexes on the

subgenomic promoter), one might expect a recombinant

ge-nome containing the nsP coding region from one virus gege-nome

and an STR coding region from another.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant AI26763.

We thank C. M. Rice for Toto:ts 21 A2 and the SIN virologists at

Washington University for helpful discussions.

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Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines

on November 9, 2019 by guest

http://jvi.asm.org/

Figure

FIG. 1. Properties of RNA II that are determined by the subgenomic pro-moter. (A) BHK-21 cells were infected with the appropriate virus at an MOI of
FIG. 2. Appearance of RNA II in different vertebrate (A) and invertebrate(B) cells, both early and late in infection
TABLE 1. Relative molar ratios of RNA II in TCS and Toto1000-infected BHK-21 cells at 37°C
FIG. 4. Sequence determination of the 3�reads from left to right (G, A, T, and C, respectively)
+2

References

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