Determinants of Dengue Virus NS4A Protein Oligomerization

Abstract

Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication. Many flaviriruses are significant human pathogens, including the four serotypes of dengue virus (DENV). DENV infection causes an acute and systemic disease that may often be asymptomatic but can lead to clinical symptoms ranging from fever to lifethreatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (1, 2). DENV is a global public health threat, with an estimated 390 million human infections per year, of which 96 million show clinical manifestations (3). Prior infection with a particular DENV serotype does not always protect against the other three serotypes (1) and is often associated with increased disease severity in the second infection (4). There are currently no licensed vaccines or antiviral drugs for either prophylaxis or treatment of DENV infection. In light of this lack of weaponry in our fight against DENV, there is an ongoing urgent need for the development of effective antivirals against the virus. Understanding the molecular details of viral replication will greatly facilitate vaccine and therapeutic development.DENV is a positive-strand RNA virus with a genome approximately 11 kb in length. The viral genome has one open reading frame flanked by 5= and 3= untranslated ...

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“…These observations suggest that RTN3.1A may interact with NS4A within the ER membrane, thus protecting NS4A from subsequent ER-associated degradation. One possible explanation is that RTN3.1A aids in the oligomerization of NS4A, as some recent reports have indicated that the oligomerization of DENV-2 NGC NS4A is mediated via the first TM domain (Lee et al, 2015;Stern et al, 2013), the domain potentially responsible for RTN3.1A-NS4A interaction. We are currently exploring this possibility.…”

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