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A PLPPV sequence in the p8 region of Gag provides late domain function for mouse mammary tumor virus.

ABSTRACT: The late (L) domain sequence used by mouse mammary tumor virus (MMTV) remains undefined. Similar to other L domain-containing proteins, MMTV p8 and p14NC proteins are monoubiquitinated, suggesting L domain function. Site-directed mutagenesis of p8, PLPPV, and p14NC, PLPPL, sequences in MMTV Gag revealed a requirement only for the PLPPV sequence in virion release in a position-dependent manner. Electron microscopy of a defective Gag mutant confirmed an L domain budding defect morphology. The equine infectious anemia virus (EIAV) YPDL core L domain sequence and PLPPV provided L domain function in reciprocal MMTV and EIAV Gag exchange mutants, respectively. Alanine scanning of the PLPPV sequence revealed a strict requirement for the valine residue but only minor requirements for any one of the other residues. Thus, PLPPV provides MMTV L domain function, representing a fourth type of retroviral L domain that enables MMTV Gag proteins to co-opt cellular budding pathways for release.

SUBMITTER: Coren LV

PROVIDER: S-EPMC6952571 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A PLPPV sequence in the p8 region of Gag provides late domain function for mouse mammary tumor virus.

Coren Lori V LV Nagashima Kunio K Ott David E DE

Virology 20190719

The late (L) domain sequence used by mouse mammary tumor virus (MMTV) remains undefined. Similar to other L domain-containing proteins, MMTV p8 and p14<sup>NC</sup> proteins are monoubiquitinated, suggesting L domain function. Site-directed mutagenesis of p8, PLPPV, and p14<sup>NC</sup>, PLPPL, sequences in MMTV Gag revealed a requirement only for the PLPPV sequence in virion release in a position-dependent manner. Electron microscopy of a defective Gag mutant confirmed an L domain budding defec ...[more]

PMID: 31357166

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Project description:The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.

Dataset Information

A PLPPV sequence in the p8 region of Gag provides late domain function for mouse mammary tumor virus.

Publications

A PLPPV sequence in the p8 region of Gag provides late domain function for mouse mammary tumor virus.

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