Genetic footprinting of a retroviral Gag gene suggests an important role in virus replication

Aretrovirus particle is a membrane-bounded sphere ≈120 nm in diameter. The principal constituent of such a particle is the Gag protein, and expression of this protein in a eukaryotic cell, even in the absence of other viral components, leads to assembly and release of virus-like particles. After release of the particle, the Gag protein molecules are cleaved by the viral protease (PR) into a series of cleavage products; this set of cleavages, collectively termed “maturation” of the particle, must occur before the particle becomes infectious. Gag proteins are always cleaved into at least three products, termed (from N to C terminus) matrix (MA), capsid (CA), and nucleocapsid (NC); additional cleavage products are frequently formed, but these are not conserved between different retroviral genera.

Although it is obvious that Gag plays a critical “structural” role in particle formation, it is now becoming clear that its cleavage products also perform important functions when a mature particle infects a new host cell. This is one of the principal implications of a new mutational analysis by Auerbach et al. (ref. 1, which appeared in a recent issue of PNAS) of an ≈600-nt stretch of the Gag coding region of Moloney murine leukemia virus (MLV), a prototype member of the Gammaretrovirus genus of retroviruses. The article presents a technical tour de force: a scan of hundreds of positions within this stretch, testing whether insertion of a 12-aa sequence at any given position interferes with the ability of the virus to replicate.

The basic strategy used in this study was developed by Singh et al. (2) several years ago. A large pool of mutants is first prepared by incubating the target DNA with an appropriate double-stranded oligonucleotide and MLV integrase. The integrase inserts the oligonucleotide, leading to the addition of 36 bases to the …