Arenavirus nucleoproteins prevent activation of nuclear factor kappa B.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses.

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.

A tetravalent recombinant dengue domain III protein vaccine stimulates neutralizing and enhancing antibodies in mice.

Dengue viruses co-circulate as four serologically distinct viruses (DENV1-4) that commonly infect individuals sequentially. Current DENV candidate vaccines incorporate the entire virion envelope E protein (E) ectodomain thereby stimulating both DENV serotype-specific and cross-reactive antibodies. Because the latter may enhance naturally acquired infection, such vaccine formulations must be tetravalent. We evaluated the neutralizing and enhancing antibody response to E domain III (dIII) proteins, in which serotype-specific neutralizing determinants are concentrated. Mice immunized with insect cell-secreted recombinant DENV-dIII proteins individually, and in tetravalent combination, produced serotype-specific IgG1 neutralizing antibodies that nevertheless exhibited measurable DENV enhancing activity in FcγR-bearing cells. Vaccine strategies directed to DENV-dIII-targeted neutralizing antibody production remain attractive but will likely require further modifications to induce safe, protective immunity.

Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype.

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.

An automated Dengue virus microneutralization plaque assay performed in human Fc{gamma} receptor-expressing CV-1 cells.

We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI-SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcgammaRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection.

Difference between the abilities of human Fcgamma receptor-expressing CV-1 cells to neutralize American and Asian genotypes of dengue virus 2.

Sera from patients involved in a Peruvian outbreak of dengue virus serotype 1 infection cross-neutralized the American genotype of dengue virus serotype 2 up to 100-fold more efficiently than they did the virulent Asian genotype of dengue virus serotype 2, as determined by a plaque reduction neutralization test (PRNT) with CV-1 fibroblasts modified to express human Fcgamma receptor CD32. The concordant preferential immune enhancement of the Asian genotype of dengue virus serotype 2 in human monocytes suggests that such a modification might strengthen the correlation between the PRNT titer and protection.

Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells.

A better understanding of the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome requires the precise identification of dengue virus (DV) permissive target cells. To examine the relative DV permissiveness among cell subsets, we inoculated unfractionated human peripheral blood mononuclear cells with DV2-16681 in the presence or absence of pooled DV-immune human sera (PHS), and assessed infection with fluorescent dye labeled DV-specific monoclonal antibody and cell surface markers using flow cytometry. We found significantly higher levels of DV antigen staining on DV-infected than mock-infected primary monocytes (3.54 +/- 3.42% vs. 0.50 +/- 0.38%; P = 0.001). The magnitude of infection was markedly enhanced in the presence of highly diluted PHS (10.04 +/- 6.10% vs. 3.54 +/- 3.42%; P = 0.015). Under identical experimental conditions, primary T or B cells were not infected either with or without the addition of PHS (0.06 +/- 0.04% and 0.44 +/- 0.22% for T and B cells, respectively). Furthermore, depletion of CD14+ monocytes prior to DV inoculation abrogated the detection of infected cells, and the addition of monoclonal antibodies to either FcgammaRI (CD64) or FcgammaRII (CD32) led to a 50-70% reduction in antibody-dependent enhancement (ADE) of DV infection. Collectively, these results provide further support to the notion that primary monocytes and FcgammaRs expressed on these cells may be important in the initial steps of immune enhancement observed in some patients with natural DV infection. They also demonstrate that using modern experimental technology, DV infection, and neutralization and enhancement of DV infection can be easily assessed simultaneously in multiple cell types.

Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32).

Fcgamma receptor (FcgammaR)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. FcgammaRIA (CD64) and FcgammaRIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory gamma-chain subunit that associates with FcgammaRIA and constitutively in FcgammaRIIA is required for phagocytosis mediated by these receptors. To determine whether FcgammaRIA and FcgammaRIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that FcgammaRIIA appeared to do so far more effectively. Abrogation of FcgammaRIA signaling competency, either by expression without gamma-chain or by coexpression with gamma-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcgammaRIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between FcgammaRIA and FcgammaRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcgammaRs.