Uncoupling cis -Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation

Abstract

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5=CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cells. D engue virus (DENV) is a member of the genus Flavivirus in the familyFlaviviridae, together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). DENV is the most significant mosquito-borne human viral pathogen worldwide, infecting more than 50 million people each year (41). Despite the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable.DENV has a plus-stranded RNA genome of about 11 kb. The single open reading frame encodes a polyprotein that is co-and posttranslationally processed by host and viral proteases. This processing yields three structural proteins (C, prM, and E) and at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (22). The coding sequence is flanked by highly structured 5= and 3= untranslated regions (UTRs) (11). In recent years, a number of cis-acting RNA elements that modulate viral RNA amplification have been identified in the viral genome (for a review, see reference 38). Viral RNA synthesis is catalyzed by the RNA-dependent RNA-polymerase activity of the viral protein NS5 and requires interaction with a 5=-end promoter element (9, 10, 13). During flavivirus morphogenesis, the newly synthesized viral RNA is r...

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

“…High-resolution reconstructions of DENV suggest that the C protein is disordered within the virus particle (6,7). Flavivirus C proteins demonstrate remarkable plasticity and are able to tolerate large deletions while maintaining protein integrity and the ability to produce viable virus (12,13,19,(78)(79)(80)(81)(82). Notably, deletions in the N termini of DENV C proteins have been shown to affect virus particle formation (13).…”

“…Flavivirus C proteins demonstrate remarkable plasticity and are able to tolerate large deletions while maintaining protein integrity and the ability to produce viable virus (12,13,19,(78)(79)(80)(81)(82). Notably, deletions in the N termini of DENV C proteins have been shown to affect virus particle formation (13). In addition, it has recently been shown that amino acid substitutions in the capsid protein of West Nile virus (WNV) can influence the virus' sensitivity to acidotropic compounds, potentially influencing virus infectivity (83).…”

“…The C protein binds nucleic acids promiscuously; how the DENV genome is targeted to sites of capsid assembly remains an area of active research (10,11). Although the amino terminus of the C protein is not required for protein integrity (6,12), deletions in the region can result in defects in particle formation in human cells (13). During virus assembly, the carboxy terminus is cleaved off the immature C protein by the viral NS2B-NS3 protease, resulting in the mature form of the protein.…”

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

“…Fundamental differences in biological processes and biochemical machineries between human and mosquito cells, such as glycosylation, membrane composition and lipid metabolism, innate antiviral responses, and other processes subverted by the virus during infection, impose pressure on viral adaptation during host switching. Differences in DENV protein processing, encapsidation, and virion maturation in mosquito and mammalian cells have been reported (1)(2)(3)(4)(5). For example, the N terminus of the capsid protein is crucial for viral encapsidation in mammalian cells but not in mosquito cells (5).…”

“…Differences in DENV protein processing, encapsidation, and virion maturation in mosquito and mammalian cells have been reported (1)(2)(3)(4)(5). For example, the N terminus of the capsid protein is crucial for viral encapsidation in mammalian cells but not in mosquito cells (5). In addition, point mutations in NS4B have been reported to decrease viral replication in mosquito cells, while the same mutations have been shown to enhance replication in mammalian cells (2).…”