It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

No abstract

Microtubules from suspension cultures of HeLa cells have been purified by carrying them through four complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C. These microtubules show, in addition to the major alpha- and beta-tubulin components, major proteins with molecular weights of 201 000-206 000 (comprising 4.5% of the total protein), proteins with molecular weights of 97 000, 100 000, 104 000, and 114 000 (together comprising approximately 2% of the total protein), and minor components with molecular weights of 68 000 and 151 000. HeLa microtubules have also been reconstituted from purified HeLa tubulin and proteins from HeLa microtubules separated from tubulin by DEAE-cellulose column chromatography. Experiments on the fractionation and reconstitution of both two- and four-cycle microtubules suggest that the 201 000-206 000-dalton proteins are incorporated into microtubules and promote tubulin polymerization. Microtubules formed by fractionationand reconstitution of two-cycle microtubules also contain several other proteins with molecular weights of 132 000, 146 000, 151 000, 160 000, and 284 000, although these are not present in microtubules carried through four assembly-disassembly cycles. Evidence is also presented which shows that a 68 000-dalton protein which is a prominent component of HeLa microtubules after two polymerization-depolymerization cycles does not stoichiometrically copurify with tubulin through repeated assembly--disassembly cycles and does not stimulate tubulin polymerization. On the other hand, the sedimentation of this 68 000-dalton protein is apparently influenced by the presence of polymerized microtubules, suggesting that this protein may be a component of a system whjich interacts weakly with microtubules. Finally, evidence is presented suggesting that two-cycle microtubules contain a proteolytic activity that can digest the 201 000-206 000-dalton proteins.

Disruption of Rauscher leukemia virus (RLV) with low levels of Nonidet P-40 yielded "immature" cores. These cores have a diameter of about 920 A, as opposed to the 1300-A diameter of RLV, possess knob-like protuberances, and contain a concentrically coiled internal strand apposed to the core shell. The two major polypeptide components of immature cores are (i) p30, the 30,000-dalton group-specific antigen, and (ii) a polypeptide that has the size and antigenic characteristics of P70, the 70,000-dalton precursor protein of the group-specific antigens of murine leukemia virus. Disruption of RLV at high ratios of Nonidet P-40 to virus yielded "mature" cores. These cores have an average diameter of 850 A, a smooth proteinaceous perimeter, and a collapsed internal strand, and they contain predominantly p30. Treatment of RLV with low levels of Nonidet P-40 for 16 hr at 22 degrees yielded cores that showed (I) a 70% decrease in the number of immature forms and concomitant increase in the number of mature forms, (II) a 60-90% decrease of P70, and (iii) a 30% increase in a 40,000- to 42,000-dalton protein. These results suggest that maturation of RLV cores is accomplished by cleavage of P70.