To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N‐terminal glycine codon (Gly‐‐‐‐Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100‐120 nm virus‐like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.
In plants, the proton pump‐ATPase (H(+)‐ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)‐ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)‐ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C‐terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild‐type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2‐ to 3‐fold increase of H(+)‐pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)‐ATPase improve the coupling between H(+)‐pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild‐type PMA2, indicating a relationship between H(+)‐ATPase activity and perturbations of the secretory pathway.
The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.
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