Dengue Virus (DV) Replication in Monocyte-Derived Macrophages Is Not Affected by Tumor Necrosis Factor Alpha (TNF-α), and DV Infection Induces Altered Responsiveness to TNF-α Stimulation

Abstract

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.

Dengue Virus Infection Induces Upregulation of GRP78, Which Acts To Chaperone Viral Antigen Production

“…on May 10, 2018 by guest http://jvi.asm.org/ assay, and the cells were lysed and RNA was extracted for analysis of DENV negative-strand RNA, which is a marker of active DENV replication (46). The results showed a reproducible 10-to 100-fold decrease in production of infectious DENV (Fig.…”

“…Cells were infected at a multiplicity of infection (MOI) of 1 to 5 for 90 min at 37°C, as described previously (34,46). Control mock infections were performed with an equal volume of heat-inactivated virus (80°C for 20 min).…”

“…Known amounts of in vitro-transcribed DENV negative-strand RNA was reverse transcribed in parallel. The tagged DENV cDNA was then subjected to real-time PCR with Quantitect SYBER Green PCR mix (Qiagen) and 0.5 M of each primer, tag, and DENV3.2, as previously described (46). The data were analyzed using RotorGene DNA sample analysis system software version 4.6.…”

“…The data were analyzed using RotorGene DNA sample analysis system software version 4.6. The real-time RT-PCR was normalized against cyclophilin A mRNA, which was reverse transcribed from total RNA as described above with 0.5 g oligo(dT) 15 (Promega), and real-time PCR was performed using primers cyclophilin (F) and cyclophilin (R), as previously described (46).…”

Dengue virus infection promotes translocation of high mobility group box 1 protein from the nucleus to the cytosol in dendritic cells, upregulates cytokine production and modulates virus replication

“…Neutralizing antibodies against gamma interferon (IFN-c), one of the cytokines that induces HMGB1, have been shown to significantly reduce circulating levels of HMGB1 in septic rats and consequently to rescue rats from lethal sepsis (Yin et al, 2005). Other agents such as glycyrrhizin, ethyl pyruvate, stearoyl lysophosphatidylcholine or nicotine have also been shown to dose-dependently inhibit HMGB1 release, thereby conferring protection against lethal sepsis (Mollica et al 2007;Chen et al, 2005;Ulloa et al, 2002;Wati et al, 2007).…”