The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10-to 100-fold decrease in infectiousvirus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.The pathogenesis of dengue virus (DENV) disease is multifactorial, and many of the clinical manifestations of the disease, including the life-threatening DENV-induced hemorrhage, may be mediated by the host responses to infection. Many studies have defined altered host responses during DENV infection, including activation of T cells (19) and altered levels of circulating factors in patients (13) and altered release of cytokines and chemokines from DENV-infected cells (8). Transcriptome analyses of DENV-infected endothelial cells (23, 45), HepG2 cells (9), circulating patient cells (39), or peripheral blood mononuclear cells from DENV-infected macaque monkeys (37) have identified alterations in transcripts involved in a variety of cellular processes, including the innate immune response, cell signaling, and metabolic processes. A recent study examined proteomic changes in DENVinfected HepG2 cells and identified 17 altered cellular proteins, including 2 proteins for which the changes were confirmed (32). In the current study, we performed a proteomic analysis of changes due to DENV infection in a target cell population that is relevant to a natural DENV infection and in which DENV-induced cytopathic effect (CPE) and the cellular death response are absent. Two-dimensional gel electrophoresis (2DGE) identified upregulation of glucose-regulated protein 78 (GRP78), otherwise known a...

Tumor necrosis factor alpha (TNF-α) is believed to play a significant role in the pathogenesis of dengue virus (DV) infection, with elevated levels of TNF-α in the sera of DV-infected patients paralleling the severity of disease and TNF-α release being coincident with the peak of DV production from infected monocyte-derived macrophages (MDM) in vitro. Since macrophages are a primary cell target in vivo for DV infection, we investigated the potential antiviral role of TNF-α in regulating DV replication in MDM. While pretreatment of MDM with TNF-α had a minor inhibitory effect, addition of TNF-α to MDM with established DV infection had no effect on DV replication as measured by DV RNA levels or progeny virus production. Blocking endogenous TNF-α using short interfering RNA or inhibitory TNF-α antibodies also had no effect on infectious DV production or viral RNA synthesis. Together, these results demonstrate that DV replication in MDM is not affected by TNF-α. Additionally, normal cellular TNF-α signaling, measured by quantitation of TNF-α-induced stimulation of transcription from an NF-κB-responsive reporter plasmid or NF-κB protein nuclear translocation, was blocked in DV-infected MDM and Huh7 cells. Thus, DV replication in MDM is not affected by TNF-α, and infected cells do not respond normally to TNF-α stimulation. It is therefore unlikely that the increased production of TNF-α seen in DV infection directly effects DV clearance by reducing DV replication, and the ability of DV to alter TNF-α responsiveness highlights another example of viral subversion of cellular functions.