RESUMO
Retroviral gag proteins, as well as fragments minimally containing the capsid (CA) and nucleocapsid (NC) subunits of Gag, are able to spontaneously assemble into virus-like particles (VLPs). This occurs in mammalian and bacterial cells as well as in in vitro systems. In every circumstance, nucleic acids are incorporated into the forming particles. Here, we took advantage of an in vitro system for the generation of non-enveloped Mason-Pfizer monkey virus (M-PMV) VLPs derived from a self-assembling CA-NC subunit of Gag. These VLPs were modified through N-terminal extension of CA-NC with short oligopeptides that, after the assembly process, were exposed on the surface of VLPs. The employed N-terminal modifications allowed specific interaction with target cells expressing prostate-specific membrane antigen. Using this system, we were able to incorporate selected siRNA into forming VLPs and deliver it into the cytosol of target cells. In comparison with other viral vectors designed for targeted transgene delivery, this M-PMV VLP system represents the lowest risk of generating virus-associated pathology, as the VLPs do not contain any viral coding sequences and are formed in a cell-free system.
Assuntos
Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Substâncias Macromoleculares/metabolismo , Vírus dos Macacos de Mason-Pfizer/genética , Transdução Genética , Virossomos/genética , Virossomos/metabolismo , Ligação Viral , Linhagem Celular , Humanos , RNA Interferente Pequeno/metabolismoRESUMO
Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.
