Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis.

BACKGROUND: Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication. How NS1 modulates infection remains uncertain given that the viral RNA replication complex localizes to the cytosolic face of the endoplasmic reticulum. METHODS AND RESULTS: Using a trans-complementation assay, we show that viruses deleted for NS1 (∆-NS1) can be rescued by transgenic expression of NS1 from West Nile virus (WNV) or heterologous flaviviruses in the absence of adaptive mutations. In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex. CONCLUSIONS: WNV RNA lacking intact NS1 genes was efficiently translated but failed to form canonical replication complexes at early times after infection, which resulted in an inability to replicate viral RNA. These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.

Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus.

Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.

Complex phenotypes in mosquitoes and mice associated with neutralization escape of a Dengue virus type 1 monoclonal antibody.

DENV1-E106 is a monoclonal antibody (MAb) with strong neutralizing activity against all five DENV-1 genotypes and therapeutic activity in mice. Here, we evaluated the potential for DENV-1 to escape neutralization by DENV1-E106. A single mutation in domain III of the envelope protein (T329A) emerged, which conferred resistance to DENV1-E106. However, the T329A variant virus had differing phenotypes in vitro and in vivo with attenuation in cell culture yet increased infectivity in Aedes aegypti mosquitoes. Mice infected with this T329A variant still were protected against lethal infection by DENV1-E106 even though much of the neutralizing activity was lost. This study reveals the complex dynamics of neutralization escape of an inhibitory MAb against DENV, and suggests that evaluation of therapeutic MAbs requires detailed investigation in relevant hosts.

2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members.

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

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.

Antagonism of the complement component C4 by flavivirus nonstructural protein NS1.

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.

Contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection.

Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.

The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32P-orthophosphate and 3H-leucine identified a 3.5-fold increase in the 32P:3H counts per minute (cpm) ratio of N in the virion as compared to the 32P:3H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32P:3H cpm ratio of N from the virion was 10.5-fold greater than the 32P:3H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication.

Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.