Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins final sigma3 and mu1/mu1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound final sigma3 protein was shown to be a poor substrate for cleavage by cathepsin D in vitro, consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions.

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE.None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2 regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin) which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE).1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. 1-5).The rod photoreceptor PDE holoenzyme is a heterotetramer consisting of two non-identical catalytic subunits (␣ and ␤) and two small subunits (␥) that serve to inhibit catalytic activity. Cone photoreceptors have a highly homologous PDE that consists of two identical catalytic subunits (␣Ј) and two conespecific ␥Ј subunits. In addition to containing the active site for cGMP hydrolysis, the catalytic subunits of rod and cone PDEs also possess noncatalytic cGMP binding sites (6). Two other classes of PDEs, namely the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5), are also known to contain noncatalytic binding sites for cGMP. In the case of PDE2, binding of cGMP at the noncatalytic site allosterically activates cyclic nucleotide hydrolysis at the active site (7,8). For PDE5, the...