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.
Highlights
Dengue virus (DENV) is the etiologic agent of the mosquito-borne diseases dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, which globally affect67–136 million people each year [1,2]
Nonstructural protein 1 (NS1) protein were detected in DENVinfected cell lysates from all cell types immunoprecipitated with anti-NS1 antibodies as compared with those immunoprecipitated with isotype-matched control antibodies, or mock-infected cell lysates immunoprecipitated with either anti-NS1 antibodies or control antibodies (Figure 1A)
Using LC-Mass spectra (MS)/MS analysis, we identified 24 potential phosphorylation sites on both cell-associated and extracellular NS1 proteins from three mammalian cell lines infected with DENV, and some of them (T27, T29, Y32, T230, and T233) were detectable at high frequency and are highly conserved among all four serotypes of DENV
Summary
Dengue virus (DENV) is the etiologic agent of the mosquito-borne diseases dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, which globally affect67–136 million people each year [1,2]. DENV is an enveloped, positive-sense, single-stranded RNA virus in the genus Flavivirus of the family Flaviviridae that includes four antigenically distinct serotypes [9]. Infection of target cells with DENV results in the production of three viral structural proteins (capsid, C; pre-membrane, prM; and envelope, E) that are required for virion formation, and seven viral nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that play important roles in viral polyprotein processing and viral RNA replication [10]. Of all of the DENV proteins, NS1 appears to be the viral protein that is detectable in the blood circulation of infected individuals at levels likely related to disease severity, so it is an important diagnostic marker of DENV infection [11,12,13].
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