A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.West Nile virus (WNV) is a single-stranded, positive-sense enveloped RNA Flavivirus that cycles in nature between birds and Culex mosquitoes. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks occur annually in North America. More than 29,000 human cases of severe WNV infection have been diagnosed in the United States since its entry in 1999, and millions have been infected and remain undiagnosed (9). Humans can develop a febrile illness that progresses to a flaccid paralysis, meningitis, or encephalitis syndrome (59). Dengue virus (DENV) is a genetically related flavivirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes clinical syndromes in humans, ranging from an acute self-limited febrile illness (dengue fever DF]) to a severe and life-threatening vascular leakage and bleeding diathesis (dengue hemorrhagic fever/dengue shock syndrome DHF/DSS]). Globally, DENV causes an estimated 50 million infections annually, resulting in 500,000 hospitalizations and ∼22,000 deaths (45).The ∼10.7-kb Flavivirus RNA genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus- and host-encoded proteases (39). The multifunctional NS proteins include an RNA-dependent RNA polymerase and methyltransferase (NS5), a helicase and protease (NS3), accessory proteins that form part of the viral replication complex, and immune evasion molecules (33, 34). Flavivirus NS1 is a 48-kDa nonstructural glycoprotein with two or three N-linked glycans, depending on the flavivirus, and is absent from the virion. The Japanese encephalitis virus (JEV) serogroup (West Nile, Japanese, Murray Valley, and St. Louis encephalitis viruses) generate NS1 and NS1′ proteins, the latter of which is a product of a ribosomal frameshift event that occurs at a heptanucleotide motif located at the beginning of the NS2A gene (25, 47).NS1 is an essential gene as it is required for efficient viral RNA replication (34, 41, 44). In infected mammalian cells, NS1 is synthesized as a soluble monomer, dimerizes after posttranslational modification in the lumen of the endoplasmic reticulum (ER), and accumulates extracellularly as higher-order oligomers, including hexamers (16, 26, 64, 65). Soluble NS1 binds back to the plasma membrane of uninfected cells through interactions with sulfated glycosaminoglycans (5). In infected cells, NS1 is also directly transported to and expressed on the plasma membrane although it lacks a transmembrane domain or canonical targeting motif. The mechanism of cell surface expression of flavivirus NS1 in infected cells remains uncertain although some fraction may be linked through an atypical glycosyl-phosphatidylinositol anchor (30, 50) or lipid rafts (49).NS1 has been implicated in having pathogenic consequences in flavivirus infection. The high levels of NS1 in the serum of DENV-infected patients correlate with severe disease (4, 37). NS1 has been proposed to facilitate immune complex formation (4), elicit auto-antibodies that react with host matrix proteins (21), damage endothelial cells via antibody-dependent complement-mediated cytolysis (38), or directly enhance infection (1). Flavivirus NS1 also has direct immune evasion functions and antagonizes complement activation on cell surfaces and in solution. WNV NS1 attenuates the alternative pathway of complement activation by binding the complement-regulatory protein factor H (11, 36), and DENV, WNV, and YFV NS1 proteins bind C1s and C4 in a complex to promote efficient degradation of C4 to C4b (3).Although NS1 is absent from the virion, antibodies against it can protect against infection in vivo. Immunization with purified NS1 or passive administration of some anti-WNV, anti-yellow fever virus (YFV), and anti-DENV NS1 monoclonal antibodies (MAbs) protect mice against lethal virus challenge (12, 13, 17, 22, 27, 29, 31, 32, 56-58). Initial studies with isotype switch variants and F(ab′)₂ fragments of anti-YFV NS1 MAbs suggested that the Fc region of anti-NS1 MAbs was required for protection (58). Subsequent mechanistic studies with WNV NS1 indicated that only MAbs recognizing cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of infected cells (13).In this study, we identify a reciprocal relationship between the secretion and cell surface expression patterns of WNV and DENV NS1s. Using WNV-DENV NS1 chimeras and point mutants, we identified a novel short peptide motif immediately C-terminal to the signal sequence cleavage position that directs NS1 for secretion or to the plasma membrane. These studies begin to explain how NS1 regulates its localization to several cellular compartments (ER, cell surface, and extracellular space) and have implications for viral infectivity, association with the host cell sorting machinery, and protective immune responses.