Vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment

The use of two-dimensional gel electrophoresis has identified the gene products A14L (p16) and A13L (p8) as abundant membrane proteins of the first infectious form of vaccinia virus, the intracellular mature virus (LMV; O. N. Jensen, T, Houthaeve, A. Shevchenko, S, Cudmore, T, Ashford, M. Mann, G, Griffiths, J, Krijnse Locker, J, Virol, 70:7485-7497, 1996), In this study, these two proteins were characterized in detail, In infected cells, both proteins localize not only to the viral membranes but also to tubular cisternal membranes of the intermediate compartment, defined by the use of antibodies to either rab1A or p21, which colocalize with rab1A (J, Krijnse Locker. S, Schleich, D, Rodriguez, B. Goud, E, J. Snijder, and G, Griffiths, J, Biol, Chem. 271:14950-14958, 1996), Both proteins appear to reach this destination via cotranslational insertion into the rough endoplasmic reticulum, as shown by in vitro translation and translocation experiments, Whereas p16 probably spans the membrane twice, p8 is inserted into the membrane by means of its single NH2-terminal hydrophobic domain, adopting a topology which leaves the C terminus exposed to the cytoplasm. Combined immunocytochemical and biochemical data show that p16 is a member of the inner of the two IMV membrane layers, whereas p8 localizes to both the inner and the outer membrane, These findings are discussed with respect to our model of IMV membrane assembly.