RESUMEN
Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.
Asunto(s)
Regulación Viral de la Expresión Génica , ARN Mensajero/biosíntesis , Rubulavirus/crecimiento & desarrollo , Transcripción Genética , Proteínas Virales de Fusión/genética , Proteínas de la Matriz Viral/genética , Animales , Línea Celular , ADN Complementario , Mutación , Plásmidos/genética , ARN Viral/biosíntesis , Rubulavirus/genética , Rubulavirus/metabolismo , Proteínas Virales de Fusión/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensayo de Placa Viral , Proteínas Virales/metabolismo , Virión/metabolismoRESUMEN
The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of approximately 250-290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH-HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20-30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.
Asunto(s)
Genes Virales/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Respirovirus/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Línea Celular , Perros , Variación Genética/genética , Genoma Viral , Modelos Genéticos , Poli A/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moldes Genéticos , Regiones Terminadoras Genéticas/genéticaRESUMEN
A dicistronic minigenome containing the M-F gene junction was used to determine the role of the simian virus 5 (SV5) intergenic regions in transcription. The M-F junction differs from the other SV5 junctions by having a short M gene end U tract of only four residues (U4 tract) and a 22-base M-F intergenic sequence between the M gene end and F gene start site. Replacing the 22-base M-F intergenic region with nonviral sequences resulted in a minigenome template (Rep 22) that was defective in termination at the end of the M gene. Efficient M gene termination could be restored to the mutant Rep 22 template in either of two ways: by increasing the U tract length from four to six residues or by restoring a G residue immediately downstream of the wild-type (WT) U4 tract. In a dicistronic SH-HN minigenome, a U4-G combination was functionally equivalent to the naturally occurring SH U6-A gene end in directing SH transcription termination. In addition to affecting termination, the M-F intergenic region also influenced polymerase reinitiation. In the context of the WT U4-G M gene end, substituting nonviral sequences into the M-F intergenic region had a differential effect on F gene reinitiation, where some but not all nonviral sequences inhibited reinitiation. The inhibition of F gene reinitiation correlated with foreign sequences having a high C content. Deleting 6 bases or inserting 18 additional nucleotides into the middle of the 22-base M-F intergenic segment did not influence M gene termination or F gene reinitiation, indicating that M-F intergenic length per se is not a important factor modulating the SV5 polymerase activity. Our results suggest that the sequence diversity at an SV5 gene junction reflects specific combinations which may differentially affect SV5 gene expression and provide an additional level of transcriptional control beyond that which results from the distance of a gene from the 3' end promoter.
Asunto(s)
Regulación Viral de la Expresión Génica , Genes Virales , Respirovirus/genética , Regiones no Traducidas 3' , Animales , Sitios de Unión , Humanos , Transcripción Genética , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Transcription of the paramyxovirus RNA genome is thought to involve a sequential stop-start mechanism whereby monocistronic mRNAs are produced by polyadenylation and termination of 3' upstream gene followed by reinitiation at the downstream start site. For a number of paramyxoviruses, transcription across the M-F gene junction results in the synthesis of high levels of a dicistronic M-F readthrough RNA. In cells infected with the paramyxovirus SV5, 15% or less of the transcripts from the viral P, M, SH, HN, and L genes were detected as readthrough products with the 3' proximal gene. By contrast, approximately 40% of the SV5 F mRNA was detected as a dicistronic M-F transcript. A comparison of the individual SV5 gene junctions showed that elevated M-F readthrough transcription correlate with the M gene end having the shortest U tract for directing polyadenylation and a gene end sequence that differs from the consensus sequence. We have tested the hypothesis that elevated M-F readthrough transcription results from an inefficient termination signal at the end of the M gene. A reverse genetics system was established whereby SV5 transcription was reconstituted in transfected cells using cDNA-derived polymerase components and dicistronic minigenomes that encoded either the SV5 M-F or the SH-HN gene junction. Chimeric SV5 minigenomes were constructed to contain exchanges of a 10 base gene end sequence and the U tract from the M-F (approximately 40% readthrough) and SH-HN (approximately 15% readthrough) junctions. Northern blot analysis of RNA synthesized from these altered templates showed that, in the context of the M-F intergenic region, increasing the length of the M gene end U tract from four residues to six or eight U residues did not decrease M-F readthrough transcription. In contrast, chimeric minigenomes that contained the 10 base region from the end of the SH gene directed very efficient gene termination and a corresponding decrease in readthrough transcription. Mutational analysis showed that a single G to A substitution located five bases 3' to the M gene U tract was sufficient to convert the M gene end region to an efficient signal for polyadenylation-termination. These results demonstrate a role for the gene end region located immediately 3' to the U tract as a major determinant of transcription termination in the paramyxovirus genome. The possible role of M-F readthrough transcription in the paramyxovirus growth cycle is discussed.