Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein

Abstract

Dengue virus (DenV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADe) of infection. phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DenV infection. this study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DenV infection. Dengue virus (DENV; family Flaviviridae, genus Flavivirus) is an enveloped, single-stranded positive-sense RNA virus that encodes for 10 proteins (three structural and seven non-structural) 1. Transmitted to humans primarily by Aedes genus mosquitoes infection can result in a range of symptoms from mild to severe. Approximately half the world population is at risk of DENV infection 2 , with three-quarters of these residing in the Asia-Pacific region, with 1.3 billion living in ten DENV endemic countries in Southeast Asia 3. There are four serotypes of DENV, DENV 1 to DENV 4. In Thailand, only 5.8% and 4.7% of primary infections were found to be caused by infection with DENV 2 or DENV 4, whereas DENV 1 and DENV 3 caused 27.5% and 29.6% of infections, respectively. Interestingly, secondary infections were found in 91.8% and 90.6% of DENV 2 and DENV 4 infections, and in patients with the clinical presentations of DHF/DSS, DENV 2 and DENV 4 were found in 87.2% and 84.4% of cases, respectively 4. Similarly, another study reported that almost all of DHF cases caused by DENV 2 and DENV 4 were secondary infections, suggesting that DENV 2 and DENV

Potential for Protein Kinase Pharmacological Regulation in Flaviviridae Infections

“…Drugs targeting CMGC kinases have been described as antiviral candidates against several flaviviruses, as well as against HCV. In the case of DENV, different studies have highlighted the MAPK/ERK pathway as essential for replication, since DENV infection can directly activate proteins in this pathway, including JNK, p38, NTRK1, MAPKAPK5, and c-src/FYN kinases [84]. JNK and p38 kinase inhibitors were reported to significantly reduce DENV protein synthesis and viral yield in infected monocyte-derived macrophages obtained from human peripheral blood [75].…”

“…For instance, the IRE1 kinase inhibitor KIRA6 reduces viral RNA levels in ZIKV infected the human HeLa cell line [90]. DENV infection has been widely reported to be inhibited by inhibitors of different members of the group, as exemplified by treatment with pyruvate kinase PKM2 inhibitor in DENV-infected U937 cells [84], the AurKB inhibitor ZM 447439 in DENV-infected Huh-7 cells [85], and the NAK family inhibitors, sunitinib and erlotinib (AAK1 and GAK subfamilies inhibitors respectively) in DENV-infected Huh-7 cells [18]. PKR is modulated by cyclophilin A, triggering antiviral responses to inhibit HCV infection in Huh 7 cell line [119], and the PKR2 inhibitor HA1077, also known as fasudil restricted HCV replication in mice [105].…”

Enteric coronavirus PDCoV evokes a non-Warburg effect by hijacking pyruvic acid as a metabolic hub

“…PK also has crucial roles in the proliferation of other viruses. For example, DENV upregulates the level of PK phosphorylation to promote viral replication [ 56 ]. Reduction of PK activity inhibits the proliferation of HCV and hepatitis B virus by decreasing the production of ATP and other glycolytic intermediates [ 57 ].…”

Revisiting dengue virus-mosquito interactions: molecular insights into viral fitness

“…At the same time, it has to evade or suppress host restriction (or anti-viral) factors (HRFs) that would otherwise suppress or inhibit infection. The availability of genomic and proteomic tools has enabled the identification of HDFs and HRFs of DENV in mammalian cells [7–12]. These studies have collectively revealed a complex network of DENV-mammalian host interactions as well, summarized in previous reviews [13–17].…”