Localization of actin in moloney murine leukemia virus by immunoelectron microscopy

Immunoelectron microscopy was used to detect actin in wild-type (wt) Molone y murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuV gag polyprot ein. Gold immunolabeling revealed the presence of actin on the surface of d elipidized VLP and delipidized wt virus particles. Statistical evaluation o f the number of colloidal gold particles per VLP revealed a large range of Values and a prevalence of VLP with small numbers of gold particles. Labeli ng for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag pr oteins p15(MA) and p12 and p30 (CA) was abundant and was not affected by tr eatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consist ent labeling for actin than without fixatives. Negative staining or heavy m etal shadowing revealed a globular surface of delipidized VLP. Stereomicrog raphs of gold-immunolabeled VLP showed that p15gag and p12gag were associat ed with the globular projections. Delipidized VLP were also well labeled wi th antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. L abeling for actin-binding proteins moesin and ezrin was negative in both th e wt virus and the VLP. The absence of Gaussian distribution of actin in th e sample of VLP suggests that actin is not a structural protein and its pre sence in MuLV virus particles may be fortuitous. This, however, does not ru le out any possible role of actin in transport, assembly, budding, or relea se of virus particles, events which take place in the cytoplasm or at the p lasma membrane. The site of actin in VLP is discussed in relation to the pr esent knowledge of the molecular organization of the MuLV gag shell. (C) 19 99 Academic Press.