Flavivirus NS4A-induced Autophagy Protects Cells against Death and Enhances Virus Replication

Abstract

Over 2.5 billion people live in areas at high risk for Dengue and related flavivirus infections (1). Dengue hemorrhagic fever, a severe complication present in ϳ5% of cases, claims more lives annually than all other hemorrhagic fevers combined (2); much of the damage is caused by death of infected cells. The fate of infected cells depends on cell type. Although flavivirus induces apoptosis of neurons and macrophages, infected hepatocytes and epithelial cells do not die. We find that flavivirus up-regulates autophagy in MDCK 3 renal epithelial cells and other cell types and subsequently protects them. We further identify nonstructural protein NS4A of both Dengue-2 and Modoc viruses as the sole viral mediator of autophagy up-regulation and protection against death. Up-regulation of autophagosomes by either live virus infection or NS4A expression depends on PI3K and is important for replication of flavivirus in renal epithelial cells. Flaviviruses often persist in liver and kidney following the acute phase of infection, suggesting that infected cells evade destruction by the host immune response, likely by flavivirusinduced up-regulation of autophagy in these cells. In vitro infection of hepatocytes and fibroblasts leads to up-regulation in autophagy and protection against death (3, 4). More relevant, Dengue-2 viruses replicate within the hepatocyte autophagosomes (5), and inhibition of autophagy attenuates virus replication (3). We extend these findings by identifying the nonstructural viral protein NS4A as the virus-encoded protein that up-regulates autophagy and thus protects the host cell against death, providing a well protected host cell for long term replication of virus.The flavivirus genome contains 10 genes encoding an ϳ11-kDa polyprotein precursor that binds to the ER membrane after translation at the rough ER as follows: three structural proteins (capsid (core) protein, pre-membrane protein ,and envelope glycoprotein) and seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that mediate viral replication, assembly, and evasion of the host immune system. The active proteins are cleaved from the ER-bound viral polyprotein by cellular proteases signalase and furin and viral protease NS3/ 2B. NS3 is both a viral protease (with required cofactor NS2B (6)) and a viral ATP-dependent helicase (7, 8); NS5 is an RNAdependent RNA polymerase and methyltransferase (9) responsible for virus genome replication, whereas NS1 is possibly a part of the viral replication complex (10). The small hydrophobic flavivirus proteins (NS2A, NS4A, and NS4B) remain the most poorly characterized. NS2A is required for the assembly of new flavivirus virions; NS4A associates with the virus replication complex and induces ER membrane rearrangements (discussed below), and NS4B is an antagonist of interferon. Flavivirus NS4A is an ϳ16-kDa membrane-associated protein consisting of four transmembrane helices and an N-terminal cytosolic region. Once the viral genome is translated in the ER,

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B

“…15,[17][18][19][20][21] Autophagy is a degradative process that is activated in response to a variety of cellular stresses, including viral infection, to maintain cellular homeostasis. This results in a high turnover of cytoplasmic proteins and organelles, which would be hypothesized to be detrimental to the expression of viral proteins required for replication and assembly.…”

“…[11][12][13][14] Several flaviviruses, including DENV and ZIKV, potently induce autophagy, which allows for efficient viral replication and virion assembly. [15][16][17][18][19][20][21][22] Interestingly, DENV infection relies on selective autophagy of lipid droplets (lipophagy) to provide energy required for viral replication. 15 A number of other forms of selective autophagy have been described including degradation of ribosomes (ribophagy), peroxisomes (pexophagy), mitochondria (mitophagy), and ER (reticulophagy).…”

Programmed cell death 50 (and beyond)

“…It now appears that autophagy usually protects cells 5,58 in that cells that can activate autophagy withstand many types of stresses far better than cells that cannot. 59,60 Cells and viruses struggle for the control of autophagy, each for their own teleonomic purposes. 59 Often the virus stimulates autophagy in the infected cell, generating resources and staving off apoptosis until the virus reproduces.…”

“…59,60 Cells and viruses struggle for the control of autophagy, each for their own teleonomic purposes. 59 Often the virus stimulates autophagy in the infected cell, generating resources and staving off apoptosis until the virus reproduces. The protein components of autophagic and apoptotic pathways can interact: autophagy can destroy damaged mitochondria or proteins signaling endoplasmic reticulum stress before they can activate apoptosis, 61 and caspases can destroy proteins that would otherwise activate autophagy.…”

Regulation of lipid stores and metabolism by lipophagy

“…The mechanism of viral induction is unknown but has been linked to a specific viral gene, NS4A. 67 The function of virus-induced autophagy in other cell types may differ, however, as autophagy has been reported to limit dengue replication in monocytes. 68 Whether other viruses such as hepatitis B, which alter autophagic function, utilize lipophagy to support replication also remains to be determined.…”