Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

The mature flavivirus particle is comprised of a nucleocapsid core surrounded by a lipid bilayer containing the membrane (M) (derived from the precursor prM) and envelope (E) proteins. The formation of intracellular prM/E heterodimers occurs rapidly after translation and is believed to be important for the assembly and secretion of immature virus particles. In this study, the role of the His at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus lifecycle was investigated. Mutations encoding basic (Arg), non-polar (Leu and Pro) and uncharged polar (Asn, Gln and Tyr) amino acids at M39 were introduced into a DENV-2 genomic-length cDNA clone and their effects on virus replication examined. Substitution of the His with non-polar amino acids abolished virus replication whereas substitution with a basic or uncharged polar amino acids moderately (~2 log10 pfu/ml decrease in viral titre; Arg and Asn) or severely (>3.5 log10 pfu/ml decrease in viral titre; Gln and Tyr) decreased virus replication. Selected mutations were introduced into a prM-E gene cassette and transiently expressed in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations were found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations which abolished virus replication prevented secretion of prM/E complexes. The results of this study pinpoint a critical residue in the M protein that potentially plays a role in viral morphogenesis, secretion and entry.