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Specific nucleotide changes in the subgenomic promoter region influence infectivity of the sindbis virus.

Transcription of the subgenomic mRNA of Sindbis virus (SINV) is initiated at a subgenomic promoter (SP). Alignment of SINV sequences identified a 68-nucleotide conserved domain spanning −19 to +49 relative to the subgenomic mRNA start site. Nucleotide T or C is present at −18 or +49 in all known SINVs while a Sindbis-like virus XJ-160 has an A or T at a corresponding position. Our results indicate that deletion or substitution of the T at +49 decreased the activity of SP, while substituting T for A at −18 did not decrease the activity of SP or genetic stability of recombinant SINV.

subgenomic mRNA synthesis on the minus strand. The junction region spanning -19 to +5 relative to the 26S mRNA start site was identified as the minimal subgenomic mRNA promoter of SINV, which is the shortest sequence that is still active as a promoter [4] . This minimal promoter encompasses the conserved junction sequence and is active in both defective interfering genomes and in SINV, although it is approximately threeto sixfold less active than the full active -98 to +14 promoter domain [4,7] . Two elements spanning positions -40 to -20 and +6 to +14 were found to be required for full promoter activity, and they act synergistically to enhance transcription [7] . Previous results suggest that 5 ′ -proximal nucleotides relative to the 26S mRNA start site are more important for the activity of the SP of SINV. The XJ-160 (GenBank accession No. AF103728) virus was used to investigate the relationship between the promoter sequence and its activity. XJ-160 is a Sindbis-like virus isolated from a pooled sample of Anopheles mosquitoes collected in Xinjiang, China, in 1990 [8] . Our results clearly demonstrate that the minimal SINV SP encompassing -19 to +5 of the start site is not always active, and that the 3 ′ -proximal nucleotides relative to the 26S mRNA start site are also important for the activity of the SP of SINV.
An alignment of SINV genomic sequences identified a 68-nucleotide conserved domain spanning positions -19 to +49 relative to the subgenomic mRNA start site. Similar to other members of the genus Alphavirus , 19 nucleotides upstream and two downstream of the initiation site are absolutely conserved among SINVs. However, a T nucleotide is found at -18 in all known SINVs, except for the XJ-160 virus, which has an A at -18. In addition, the XJ-160 virus has a T at +49, while other SINVs have a C at +49, which is positioned next to the initiation codon (+50 to +52) of the viral structural proteins. To investigate the effects of the T nucleotide at -18 or +49 on the activity of the 26S promoter and the function of the minimal SINV promoter, several SPs (SP1 to SP5) of the XJ-160 virus were designed as shown in figure 1 . Promoter SP1 encompassing the domain -19 to +5 relative to the 26S mRNA start site is identical to the SINV minimal 26S mRNA promoter. Compared with the wild-type promoter SP2 (-19 to +49), SP3 and SP4 have an A to T change at -18 and T to C change at +49, respectively. Promoter SP5 contains a deletion of the T at position +49 relative to SP2.
Recombinant plasmid pBR-XJ160 is an infectious fulllength cDNA clone of the XJ-160 virus, from which rescued virus can be obtained by transcription in vitro and transinfection [9] . Based on pBR-XJ160, recombinant clones with two 26S promoters were constructed to investigate subgenomic mRNA promoter function: a wild-type 26S promoter for expression of the structural protein, and a designed promoter, which was placed upstream of a reporter gene ( enhanced green fluorescence protein (EGFP) or Gaussia luciferase (GLUC) ). As can be seen in figure 1 , the reporter gene cassettes consisted of a reporter gene ( EGFP or GLUC ), and the designed SPs were placed between the 3 ′ -terminus of the virus structural genes and the 3 ′ -untranslated region (3 ′ UTR). Recombinant clones carrying EGFP and SP1, SP2, SP3, SP4 or SP5 were designated pBR-XJ11, pBR-XJ12, pBR-XJ13, pBR-XJ14 or pBR-XJ15, respectively. Similarly, recombinant clones with GLUC and SP1, SP2, SP3, SP4 or SP5 were named pBR-XJ21, pBR-XJ22, pBR-XJ23, pBR-XJ24 or pBR-XJ25, respectively. Rescued viruses could be obtained by transfecting 2 μg RNA transcripts from the above recombinants into BHK-21 cells. The corresponding virus resulting from the transfection of clone pBR-XJ11 to pBR-XJ15 was designated XJ-11 to XJ-15. Similarly, the rescued virus from pBR-XJ21 to pBR-XJ25 was named XJ-21 to XJ-25.
After the transfection of BHK-21 cells with the RNA transcripts from the recombinant clones, a typical cytopathic effect similar to that induced by XJ-160 virus could be seen 24 h postinfection (h p.i.), such as cell rounding and a loss of adherence (data not shown). However, no cytopathic phenotype was observed in BHK-21 cells infected with RNA transcripts from pBR-XJ11 or pBR-XJ21 with an SINV minimal SP (-19 to +5). To further examine the effects of the designed promoter on the propagation of XJ-160, growth curves of the recombinant viruses were constructed in BHK-21 cells. As shown in figure 2 , compared with XJ-160 virus, slightly higher titers of XJ-12 or XJ-13 virus were detected at 8-48 h p.i. In contrast, lower titers of XJ-14 or XJ-15 virus were consistently detected during infection. The growth curve from XJ-21 to XJ-25 virus carrying GLUC and designed SPs had similar characteristics to that of the XJ-11 to XJ-15, respectively (data not shown). In addition, the growth tendencies of the mutant viruses were similar to that of XJ-160 virus. For evaluating the difference between the wild-type virus and mutant viruses, significant differences were performed using the oneway ANOVA analysis function in the SPSS software package (release 12.1; SPSS, Inc., Chicago, Ill., USA). Differences were considered significant at p < 0.05. These results indicate that the minimal SINV SP had no activity in XJ-160 virus, and that substitution of the T for A at -18 had no marked effect on the secretion of replicated virions, and slightly enhanced the propagation capacity of XJ-160 virus. In contrast, the differences between the mutant viruses with mutation at +40 (XJ-14 and XJ-15) and XJ-160 virus were statistically significant at all indicated time points (p < 0.05), suggesting that deletion or substitution of the T at +49 significantly decreased viral propagation capacity.
To further examine the relationship between the SP sequence and its function, reporter gene assays were performed as described previously [10] . Specific green fluorescence was observed in BHK-21 cells transfected with XJ-12 to XJ-15 virus, while no EGFP expression was observed following transfection with pBR-XJ11 ( fig. 3 a), confirming the nonfunction of the SINV minimal SP in XJ-160 virus. In BHK-21 cells infected with XJ-12 or XJ-13 virus, continuously enhanced fluorescence could be seen 24-72 h p.i.; the EGFP expression peak was found at 48 h p.i. On the passaging of XJ-12 to XJ-15, EGFP expression was observed in cells infected with repeated passages of XJ-12 or XJ-13 virus, while no specific green fluorescence was observed after XJ-14 or XJ-15 virus had been passaged three times (data not shown). Similarly, continuously high luciferase activity was detected in BHK-21 cells infected with repeated passages of XJ-22 or XJ-23 virus, while only background levels of luciferase were detected after XJ-24 or XJ-25 virus was passaged  However, passage 1 of XJ-24 or XJ-25 virus exhibited significantly higher GLUC activity than the following passages of the corresponding virus (p < 0.05). These results further demonstrate the distinct effects of the two T nucleotides in the 26S mRNA promoter on the infectivity of XJ-160 virus. For some positive-stranded RNA viruses, including picornaviruses and flaviviruses, genomic RNA is the only viral mRNA synthesized in infected cells. For many other positive-stranded RNA viruses, including alphaviruses and coronaviruses, one or more subgenomic RNAs are also synthesized in infected cells. Two distinct mech-anisms for generating subgenomic RNAs have been identified. First, subgenomic RNA synthesis by coronaviruses involves discontinuous RNA synthesis [11] . The second is the initiation of transcription at an internal site called an SP on the minus strand of the genomic RNA. The second mechanism is the most clearly established in alphaviruses [6,12,13] and the plant virus brome mosaic virus (BMV) [14,15] . Thus, binding between RNAdependent RNA polymerase (RDRP) and SP may be a major contributor to the transcription of 26S mRNA. nsP4, which has been identified as the Sindbis viral RDRP, is highly conserved in the genus Alphavirus , and plays a major role in viral RNA replication [16,17] . nsP4 also recognizes the SP, and the specific amino acid sequence in nsP4 responsible for recognizing SP is  [18] . Substitution of the T for A at -18 had little effect on the activity of the SP or the characteristics of XJ-160 virus. One explanation for this is that RDRP is able to tolerate sequence changes at this position.
Compared with substitution of the T for A at -18, change or deletion of the T at +49 resulted in very low activity levels of report genes. Since the +49 mutation is positioned next to the initiation codon of the reporter genes, it is likely that the translatability of the second mRNA is altered by the change at the position of +49. To confirm the contribution of +49 to promoter function, virus-specific RNAs synthesized by the recombinant virus both in passage 1 and 3 were analyzed as described previously by Hertz and Huang [19,20] . As shown in the figure 4 , all the mutant RNA transcripts produce a genomic and two subgenomic mRNAs at passage 1. Compared with XJ-11 with the substitution at -18, the levels of virus-specific RNAs from the RNA transcripts with the +49 mutation are obviously decreased at passage 3, including XJ-14, XJ-15, XJ-24 and XJ-25. Especially, two subgenomic mRNAs are hardly detected in the BHK-21 cells infected with RNA transcripts with the +49 mutation, suggesting that the contribution of +49 mutations to promoter function may be related with the translatability of the second mRNA.
Host-dependent evolution of SINV promoter for subgenomic mRNA synthesis and evolution of SP in cultured cells were analyzed by Hertz and Huang [19,20] , which is the first report of construction of double SP cDNA clone of SINV. The analysis of subgenomic mRNA promoter function using a full-length cDNA clone of SINV is problematic. First, the clone is somewhat cumbersome to manipulate because of its large size and lack of unique restriction sites around the junction region. The second, and more serious, impediment is that the open reading frame of the nonstructural protein nsP4 overlaps with the initiation site for subgenomic RNA synthesis. Full-length pBR-XJ160 is 14,625 bp, and no reasonable unique restriction sites for the construction of a recombinant clone with two promoters have been found. Two unique restriction sites, a Kas I site at the 3 ′ -terminal end of the E1 gene and a Xho I site at the 3 ′ terminal of the polyA tail, were used to construct the desired clones in this study. These were generated by three long-fragment fusion PCRs and two ligation reactions. In addition, the designed SPs were placed upstream of the reporter genes, which avoided the effects of nsP4 overlap on the subgenomic mRNA promoter. In this way, we constructed competent viruses expressing EGFP or GLUC, such as XJ-12, XJ-13, XJ-22 and XJ-23. These represent a powerful tool for studying the replication strategy and pathogenetic mechanisms of alphaviruses. and 3 from the cells and were denatured and separated through 1% agarose. The gel was treated for fluorography and exposed to film. The genomic RNA (big arrow) and the two subgenomic mRNAs (small arrows) were detected.