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Updated: Aug 16, 2026
Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System
Published on: July 17, 2021
Abundant authentic MMTV-Env production from a recombinant provirus lacking the major LTR promoter
Stefanie Rungaldier1, Shiva Badihi Nejad Asl, Walter H Günzburg
1Research Institute of Virology and Biomedicine, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
Abstract:
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.
Insights
Mouse mammary tumor virus (MMTV) envelope proteins can be produced without the P1 promoter. The R region acts as a TATA-less initiator, driving spliced env mRNA transcription in MMTV proviruses.
Area of Science:
- Retroviral gene expression
- Molecular virology
- Mammalian retroviruses
Background:
- The envelope (env) mRNA in Mouse Mammary Tumor Virus (MMTV) is typically a singly spliced product initiated from the P1 promoter.
- Understanding alternative transcription initiation mechanisms is crucial for retroviral replication and pathogenesis.
Purpose of the Study:
- To investigate alternative pathways for MMTV envelope protein production.
- To identify regulatory elements involved in MMTV transcription initiation.
- To characterize the role of the R region and LTR in MMTV gene expression.
Main Methods:
- Reporter assays to assess transcriptional activity.
- Transient and stable transfection of MMTV proviral constructs.
- 5' Rapid Amplification of cDNA Ends (RACE) to identify transcription start sites.
- Dexamethasone induction studies.
Main Results:
- Envelope proteins are produced inducibly even without the P1 promoter.
- The R region is essential for protein production in various MMTV contexts.
- A sequence within the R region functions as a TATA-less initiator.
- The 5' LTR's U3 region is required for R-initiator activity in integrated proviruses.
- Stable cell clones produced Env mRNA but not infectious virions in the absence of P1.
Conclusions:
- The R region can drive transcription of spliced env mRNA independently of the P1 promoter.
- Alternative transcription initiation mechanisms contribute to MMTV gene expression.
- The integrated provirus context influences the regulatory role of the LTR in MMTV transcription.
