The retroviruses are enveloped viruses, therefore during the late phase of their life cycle they need to recruit viral membranous envelope originating from the cytoplasmic membrane from the host cell. The interaction of viral immature particle with the membrane is facilitated by structural protein matrix protein (MA). Like all structural proteins, MA in immature viral particle is a part of Gag polyprotein and it is cleaved by viral protease after virus particle leaves host cell. MAs of most retroviruses are myristoylated and the myristoyl is necessary for the interaction with the membrane, since it serves as an anchor of whole Gag in phospholipid bilayer. The molecule responsible for the binding of MA to the proper membrane is phosphatidylinositol-4,5-bisphosphate (PIP), a phospholipid found exclusively in cytoplasmic membrane. It has been proven for HIV-1, that PIP significantly improves binding of retroviral MAs on the plasma membrane1,2.
There are two main types of retroviral virus particle assembly. The B/D type retroviruses assemble the immature virus particle in the cytoplasm and it is then transported towards the membrane. The C-type Gag proteins are transported directly towards the plasma membrane and the formation of immature virus particle occurs on the membrane along with the budding. This type of retrovirus must also affect the interaction of gag with the membrane, since in C-type only single molecules interact with the membrane, while in D-type it is whole immature virus particle. In this work, we used MA of Moloney Murine Leukemia Virus (MoMLV) as a C-type retrovirus model and Mouse Mammary Tumor Virus (MMTV) MA as a model of B/D type retrovirus. We measured the binding of these MA proteins with liposomes of various phospholipid compositions and compared the strength of the interaction between these two proteins and also to compared it with other retroviral MAs (mainly with HIV-1 as a C-type and Mason-Pfizer Monkey Virus as a B/D-type).
The proteins were produced in E. coli cells. The myristoylation was facilitated by co-expression of MAs with yeast N-myristoyl transferase. The purified proteins were then used for interaction with artificial liposomes composed of different lipids and different amounts of PIP. The unbound protein was subsequently removed from the liposomes by ultracentrifugation in sucrose gradient and analyzed by SDS-PAGE.
This work was supported by Czech Science Foundation grant GA17-24281S.