Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle (original) (raw)

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses. M embers of the genus Flavivirus within the family Flaviviridae comprise of several medically important pathogens including the Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV) which cause significant morbidity and mortality in humans, animals and birds 1. Flaviviruses are enveloped viruses with a positive-sense, single-strand RNA genome of approximately 11 kb that is translated as a single polyprotein precursor of ,3300 amino acids in length and proteolytically cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane (prM-M), and envelope) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins 1. Flaviviruses enter their host cells through a process of receptor-mediated endocytosis followed by subsequent fusion within the endosomal compartment to release the viral genome into the cytoplasm for translation and replication 2. Replication of the viral RNA genome occurs on virus-induced host cell membranes. Such structures may serve as a scaffold for anchoring the viral replication complexes, which consist of viral RNA, viral proteins, and host cell factors. Virus assembly occurs within the endoplasmic reticulum-derived membrane compartments and non-infectious virions traverse through the Golgi stack to reach the trans-Golgi network (TGN) where furin-mediated cleavage between prM-M leads to conformational changes rendering the virion infectious. Infectious virus is subsequently released from cells via the secretory pathway 1,3. Adaptor complexes (AP-1 through 5) are heterotetrameric protein complexes comprising one each of the two large sub-units c, a, d, e and f and b1-5 and one medium sub-unit m1-5 and one small sub-unit s1-5 respectively. APs are involved in distinct intracellular vesicular transport pathways which play a vital role in maintaining cellular homeostasis 4-6. AP-1 is involved in the trafficking of cargo molecules in the biosynthetic pathway from the trans-Golgi network (TGN) to endosomes and back. AP-2 which is one of the most extensively-studied adaptor complex has been shown to be involved in the endocytic pathway at the plasma membrane. AP-3 is reported to function in the transport of selected proteins in the endo-lysosomal pathway. AP-4 is involved in sorting of proteins destined to basolateral surface in polarized cells. AP-5 is the newest member of the family discovered recently and has been proposed to function at the late endosomes 6. Despite the prominent role played by APs in intracellular trafficking pathways, their involvement in flavivirus life-cycle has not been characterized. A number of earlier studies investigating the internalization of flaviviruses have shown the involvement of clathrin-dependent and lipid raft-dependent pathways for virus entry but the role of APs in stages post-entry has not been investigated 2. A genome-wide RNA interference screen for identifying cellular proteins associated with WNV infection identified AP-1 m1 subunit and AP-3 s2 subunit as some of the host factors required for both WNV and DENV infection 7. Similarly, another study identified AP1M1 as one of the genes required for