Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.
Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of 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. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.
National Institute for Biological Standards and Control, South Mimms, Hertfordshire, EN6 3QG, United Kingdom. mnermut@nibsc.ac.uk
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D016475:3T3 Cells; D000199:Actins / Q000648:ultrastructure*; D000818:Animals; D002460:Cell Line; D004285:Dogs; D015683:Gene Products, gag / Q000648:ultrastructure; D007150:Immunohistochemistry; D051379:Mice; D016253:Microscopy, Immunoelectron; D008979:Moloney murine leukemia virus / Q000648:ultrastructure*; D011092:Polyethylene Glycols; D014771:Virion / Q000648:ultrastructure
D000199:Actins; D015683:Gene Products, gag; C024062:nonidet; D011092:Polyethylene Glycols