Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Abstract

Temperature-sensitive mutants of Semliki Forest virus (SFV) and Sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts . When antisera against purified glycoproteins and '25 1-labeled protein A from Staphylococcus aureus were used, only small amounts of virus glycoproteins were detected at the surface of SFV is-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39°C) . When the mutant-infected cells were shifted to the permissive temperature (28°C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10p,M) and carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP; 10-20 ,AM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 jug/ml) inhibited the transport by^-50%, whereas cytochalasin B (1 l-Ag/ml) had only a marginal effect .Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39°C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28°C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum . A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of -10 min . These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.

Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively

“…First, incubation of cells with MON is thought to arrest viral glycoprotein movement within the Golgi apparatus (20,21,42,44), yet our RIP analysis after pulse-chase experiments (Fig. 4A) indicated that KUN structural proteins are still secreted under these conditions.…”

Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex.

“…The glycoproteins of SFV and VSV have been extensively used as model systems for studying the transport of eucaryotic membrane glycoproteins from the rough endoplasmic reticulum to the plasma membrane via the Golgi complex (1,7,11,16,29,31,38,39). As with SFV and VSV, the Uukuniemi virus glycoproteins were localized to the rough endoplasmic reticulum as well as to the nuclear membrane, reflecting the synthesis of viral membrane-bound glycoproteins at these sites.…”

“…Johnson and Schlesinger (10) have recently shown that when cells infected with vesicular stomatitis virus (VSV) or Sindbis virus are treated with the carboxylic ionophore monensin, the cellular location of virus maturation is markedly changed. Monensin treatment leads to the vacuolization of the Golgi apparatus (32) and causes the arrest of virus membrane proteins at this site of the cell (10,11,31).…”

Monensin inhibits Semliki Forest virus penetration into culture cells.

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. but virus maturation is inhibited because the spike glycoproteins are not transported to the cell surface (17,18 (15,19). We found that, in addition to the effect on virus maturation (17,18) (Fig.…”