RESUMO
Unique among the retroviruses, mouse mammary tumor virus (MMTV) carries, in addition to the usual long terminal repeat (LTR) promoter, another promoter, P2, which is located in the central part of the proviral U3 sequence, within the LTR open reading frame (ORF). Using an in vitro reporter system based on a sensitive luciferase expression assay, we investigated the regulation of the P2 promoter in the context of the Mtv-2 and Mtv-8 genomes. Irrespective of the genomic source, the activity of the P2 promoter is regulated by a downstream-located enhancer and an upstream-located negative regulatory element (NRE), the activity of which overrides the activator. During this study, we unexpectedly detected another independent neighboring promoter that we called P3. The novel P3 promoter does not seem to be controlled by any NRE but is influenced by the same enhancer that modulates the P2 promoter. The respective transcription starts of the two promoters located in this tight cluster are only 61 bases apart. The transcripts originating from this promoter complex carry the same first intron, which is bound by canonical splice donor and splice acceptor sites located in the LTR. One novel doubly spliced transcript carrying a 459-nucleotide-long ORF was detected in several MMTV-carrying murine cells and could be successfully expressed in murine cells as a His-tagged fusion product. The novel viral protein, the function of which remains to be elucidated, has an apparent molecular mass of 20 kDa.
Assuntos
Regulação Viral da Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes Reporter , Luciferases/análise , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Sítios de Splice de RNA , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
As for all retroviruses, the env mRNA is thought to be a singly spliced product of the full-length transcript from the P1 promoter in the MMTV provirus. However, we show that envelope proteins can be produced in an inducible manner in the absence of the P1 promoter from an otherwise complete provirus. Furthermore, we demonstrate in both reporter assays and the proviral context that the R region is necessary for protein production in transiently transfected cells and in a number of independent, stably transfected cell clones. Using 5' RACE, we show that a sequence within the R region functions as a TATA less initiator. The most distal part of the 5' LTR (first 804 bases of the U3 region) is required for the activity of the R-initiator element only when the provirus is integrated. Transfection with a full-length proviral DNA carrying a deletion of P1 in the 5' LTR resulted in the establishment of stable cell clones able to produce Env in a dexamethasone-dependent manner but not infectious virions. We therefore conclude that in the absence of P1, R can drive transcription of the spliced env mRNA but not genomic viral RNA.