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

Abstract

The mature flavivirus particle comprises 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 residue at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus life cycle 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 were examined. Substitution of the His residue with non-polar amino acids abolished virus replication, whereas substitution with basic or uncharged polar amino acids decreased virus replication moderately (~2 log 10 p.f.u. ml "1 decrease in viral titre for Arg and Asn) or severely (>3?5 log 10 p.f.u. ml "1 decrease in viral titre for Gln and Tyr). Selected mutations were introduced into a prM-E gene cassette and expressed transiently in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations was found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations that 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. INTRODUCTIONDengue virus (DENV) is transmitted by mosquitoes and causes the most important arthropod-borne viral disease of humans, with 50-100 million individuals infected annually worldwide (Gubler, 2002). The four serotypes of DENV (1-4) are members of the genus Flavivirus within the family Flaviviridae, along with a number of other medically important viruses that include Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV) and yellow fever virus (YFV) (Burke & Monath, 2001).The mature flavivirus particle contains three structural proteins, the capsid (C), envelope (E) and membrane (M) (derived from the precursor prM) proteins, plus a lipid bilayer and a single strand of infectious RNA. The structure of the mature DENV-2 particle has been determined by fitting the X-ray crystallographic structure of the TBEV E protein (Rey et al., 1995) into a 24 Å resolution cryoelectron microscopic (cryo-EM) reconstruction of the DENV-2 particle (Kuhn et al., 2002). The structure revealed that 90 E protein dimers lying parallel to the membrane in a 'herringbone' conformation and 180 M proteins form a tightly packed outer icosahedral protein shell surrounding a lipid bilayer, which encloses a disordered nucleocapsid consisting of multiple copies of the C protein surrounding the RNA genome (Kuhn et al., 2002).The flavivirus RNA genome is approximately 11 kb in size and encodes a large polyprotein with the gene order NH 2 -C-prM-E-N...

Differential Modulation of prM Cleavage, Extracellular Particle Distribution, and Virus Infectivity by Conserved Residues at Nonfurin Consensus Positions of the Dengue Virus pr-M Junction

“…Each of the remaining upper fractions contained 6.5% or less of the total E signal, with a small peak in fractions 10 to 11. Consistent with the findings of previous reports (20,34), the prM proteins in the upper sucrose fractions were enriched with the faster-migrating species, whereas those in the lower fractions contained the faster-and slower-migrating species in approximately equal amounts (Fig. 3A).…”

“…Therefore, the majority of extracellular viral particles generated by the parent virus were rapidly sedimenting virions. The predominance of virions observed for the 16681Nde(ϩ)-infected C6/36 cultures was in accordance with previous results obtained from dengue virus-infected C6/36 and Vero cells (24,34). A similar distribution of particles was observed with the E199A and S206A mutants, in which prM cleavage was minimally affected (Fig.…”

“…Among flaviviruses, the proportion of the two types of particles generated during viral infection is quite variable. The majority of particles released from dengue virus-infected Vero cells and C6/36 mosquito cells are virion-sized particles (24,34). These large particles predominate in Japanese encephalitis virus (JEV)-infected Vero cell cultures, whereas subviral particles are more abundant in cultures of infected C6/36 cells (20,23,25,29).…”

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

“…However, little is known about the role of M protein including the MH domain (positions 112-131) in the replication cycle of DENV. Previously, Pryor et al (44) reported that a histidine residue at position 130 is important for assembly of DENV2 virions; we reported that proline substitutions of residues in the MH domain (positions 120, 123, and 127) of DENV prM protein greatly affect the entry and assembly of VLPs and virions, suggesting the importance of the C terminus of MH domain (45). Sequence analysis of the MH domain revealed nine residues highly conserved among different strains of four DENV serotypes (see Fig.…”

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

“…The perimembrane and transmembrane helices serve to anchor the M protein to the membrane. In the case of DV and JEV, the perimembrane helix region was shown to be involved in virus assembly (30)(31)(32)(33) and entry (31,32). Subsequent mutagenesis analysis of the M protein proved the importance of an interactive network between E and M in those processes (16,34,35).…”