Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Abstract

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

Molecular Detection and Genetic Characterization of Japanese Encephalitis Virus in Animals from 11 Provinces in China

“…The JEV strains examined during the current study have retained this immunogenic capacity, which is good news for JEV control. Phosphorylation affects viral amplification [ 32 ], and E-protein phosphorylation sites were found to be very stable among strains. OP699290, OP699291 and OP699301 showed deletion of the 76T phosphorylation site and decreased phosphorylation at the 251S position, perhaps due to mutation of the amino acid sites.…”

Activation of Early Proinflammatory Responses by TBEV NS1 Varies between the Strains of Various Subtypes

“…Therefore, a further study of the substitutions at this position may reveal additional virulence determinants. In another study, the importance of dengue virus NS1 phosphorylation has been observed [ 45 ]. Some mutations significantly decreased the production of infectious virus but did not affect relative levels of intracellular NS1 expression or secretion.…”

“…However, the levels of IL-6 and IL-10 can underlie the differences between the strains or subtypes of TBEV. For other members of the flavivirus group, i.e., dengue virus, it has been suggested that the role of NS1 in pathogenesis is also strain-dependent [ 45 ].…”

Genomic Characteristics and E Protein Bioinformatics Analysis of JEV Isolates from South China from 2011 to 2018

“…Compared with the classical strains, the E protein of the four JEV isolates in this study was more conservative. Dechtawewat et al [39] used immunoprecipitation with specific antibodies and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the potential phosphorylation sites on the NS1 protein of dengue virus (DENV), revealing that DENV NS1 phosphorylation has a functional effect on viral amplification during DENV infection. These findings emphasize the importance of phosphorylation and can also be used as experimental evidence to verify the predicted phosphorylation sites in this study, which may pave the way for the future design of targeted specific antiviral drugs.…”