Role of a heterologous retroviral transport element in the development of genetic complementation assay for mouse mammary tumor virus (MMTV) replication

Abstract

The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

“…The potential of these RNAs to be packaged by virus particles was tested by using a 3-plasmid trans complementation assay developed earlier to study MMTV packaging and propagation [23–25]. In this assay, infectious, pseudotyped, virus particles were created by expression of the MMTV gag/pol and vesicular stomatitis virus (VSV-G) env genes from two independent plasmids, while the transfer vector described above provided the wild type or mutant substrates for RNA packaging (Supplemental Fig.…”

“…In parallel, the virus particles isolated from the transfected cultures were used to: i) measure the gRNA content in the virus particles by RT-qPCR, and ii) infect target cell line HeLaT4, resulting in the transduction of these cells with the marker hygromycin resistance gene (propagation of the packaged RNA), as described previously [23–25]. The number of hygromycin resistant (Hyg r ) colonies obtained should be directly proportional to the amount of RNA packaged into the virus particles, providing an indirect estimate of RNA packaging, given that packaged RNA is successfully reverse transcribed and integrated.…”

“…The number of hygromycin resistant (Hyg r ) colonies obtained should be directly proportional to the amount of RNA packaged into the virus particles, providing an indirect estimate of RNA packaging, given that packaged RNA is successfully reverse transcribed and integrated. Thus, this in vivo packaging and propagation assay allowed us to quantify the effects of the mutations in the SL4 region on RNA packaging and propagation without any ambiguity since the defective nature of the virions produced limited the assay to a single round of replication [23–25]. …”

“…The case of MMTV is not that different. We have recently shown that the entire 5ʹ UTR and the first 120 nt of gag are required for efficient MMTV gRNA packaging and propagation [23,24]. Furthermore, these sequences were predicted to fold into higher order structures comprising of several structural motifs, which were validated by SHAPE (selective 2ʹ-hydroxyl acylation analyzed by primer extension [25].…”

“…Therefore, to establish the biological significance of the sequences and structural components of SL4 and to further provide functional evidence for the role of pal II and ssPurines in MMTV RNA packaging, a series of mutations were introduced that included deletions, substitutions, and compensatory mutations in SL4. These mutations were tested employing a biologically relevant MMTV-based three plasmid trans complementation assay [23] to determine their direct effects on the packaging and propagation of MMTV transfer vector RNAs, and finally structure-function relationship of these mutations was established using mFold predictions and SHAPE experiments. Our results show that the bifurcated nature of SL4 structure as well as the sequence of its two apical loops are required for MMTV gRNA packaging and propagation.…”