Luteolin decreases the yield of influenza A virus in vitro by interfering with the coat protein I complex expression.

Influenza is an acute transmissible respiratory infectious disease in humans and animals with high morbidity and mortality. It was reported that luteolin, extracted from Chinese herbs, could potently inhibit influenza virus replication in vitro. To assess the effect and explore the fundamental mechanism of luteolin, we infected several cell lines with two subtypes of influenza A virus (IAV), including A/Jiangxi/312/2006 (H3N2) and A/Fort Monmouth/1/1947 (H1N1) and demonstrated that luteolin suppressed the replication of IAV by cytopathic effect reduction method, qRT-PCR, immunofluorescence and Western blot assays. A time-of-addition assay indicated that this compound interfered with viral replication at the early stage of infection. In addition, we found that luteolin suppressed coat protein I complex expression, which was related to influenza virus entry and endocytic pathway. Overall, our findings demonstrated the antiviral effect of luteolin against IAV and its novel antiviral mechanism.

Effects of picornavirus 3A Proteins on Protein Transport and GBF1-dependent COP-I recruitment.

The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.

A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1.

Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.

Inhibitors of COPI and COPII do not block PEX3-mediated peroxisome synthesis.

In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.

Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion.

Incubation of NRK cells for 30 to 45 minutes with 50 mM 2-deoxy-D-glucose (DOG) in glucose and pyruvate-free medium results in depletion of the cellular ATP pool and in specific disassembly of the cis-Golgi network (CGN), with the stack of Golgi cisternae (SGC) and the trans-Golgi network (TGN) remaining intact and sensitive to BFA. The disassembly of the CGN is mediated by long tubular structures extending outwards from the Golgi complex and involves microtubules. Upon removal of DOG and addition of glucose and pyruvate to the culture medium, the morphology of the CGN is slowly reestablished. Reconstruction of the CGN involves COPI/COPII-positive vesicles that resume the transport of proteins and in particular of CGN membrane proteins out of the ER. Exit of CGN membrane proteins from the ER is insensitive to BFA. In cells pretreated with nocodazole, the CGN membrane proteins are transported to the vicinity of the SGC fragments dispersed throughout the cytoplasm. Ultrastructural studies of cells engaged in the reconstruction of the CGN revealed that the CGN cisterna emerge as tubular structures extending from 0.2-0.3 microm uncoated vesicles prior to their organization on the cis-side of the SGC.