Biochemical and functional characterization of mouse mammary tumor virus full-length Pr77Gag expressed in prokaryotic and eukaryotic cells

Akhil Chameettachal, Vineeta Narayana Pillai, Lizna Mohamed Ali, Fathima Nuzra Nagoor Pitchai, Mustafa Taleb Ardah, Farah Mustafa, Roland Marquet, Tahir Aziz Rizvi

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.

Original languageEnglish
Article number334
JournalViruses
Volume10
Issue number6
DOIs
Publication statusPublished - Jun 18 2018

Keywords

  • Gag interactions
  • Mouse mammary tumor virus (MMTV)
  • Pr77
  • Protein assembly
  • Protein expression
  • Protein interaction
  • Protein purification
  • RNA
  • RNA
  • RNA packaging
  • Retrovirus

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

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