Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

Abstract

The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1= position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1= position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1= position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to -4) are mosquito-borne viruses of the Flavivirus genus that significantly impact public health (1, 2). Despite a clear need, neither vaccines nor therapeutics for WNV or DENV have been licensed for use in humans. The flavivirus genome is an ϳ11-kb, single-stranded, positive-sense RNA that encodes a single open reading frame flanked by 5= and 3= untranslated regions. The viral genome is translated on endoplasmic reticulum (ER)-derived membranes into a single polyprotein that undergoes co-and posttranslational cleavage by the viral protease NS2B/3 and host proteases into 10 functionally distinct proteins, including the structural proteins capsid (C), premembrane (prM), and envelope (E) that form the virus particle. During assembly, membrane-anchored prM and E glycoproteins are incorporated into virions as they bud into the ER lumen. The C protein associates with the viral genome in the cytoplasm to form an unstructured nucleocapsid that is incorporated into the budding particle via unknown mechanisms (3). The carboxy terminus (C terminus) of the C protein includes a signal sequence, flanked by protease cleavage sites, that directs the translocation of prM into the ER lumen and tethers C to the cytosolic face of the ER membrane.Cleavage at both sites is essential for virion morphogenesis and occurs in a sequenti...

Oral Vaccination with a DNA Vaccine Encoding Capsid Protein of Duck Tembusu Virus Induces Protection Immunity

“…Third, the capsid dibasic-site, which is a cleavage region in the capsid protein, is important for virus growth [ 47 ]. As a result of blocking this site, the virus particle release from infected cells can be restricted [ 48 ]. Finally, increasing evidence indicates that capsid proteins can contact many host proteins to modulate the host immune signal in the cytoplasm and nuclei [ 19 , 49 ].…”

Tick-Borne Encephalitis Virus: A Structural View

“…The cleavage of the C-terminal membrane anchor first from C and then from prM is sequential and strictly controlled. In Murray Valley encephalitis virus, YFV, and WNV, the perturbation of the cleavage order results in excessive formation of NC-deficient particles [ 96 , 97 , 98 , 99 ]. For TBEV, however, the uncoupling of these events only affects particle production in tick cells [ 97 ].…”

Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles

“…We provide an insight into the relative processing efficiency of Ca from DENV2, WNV and ZIKV in the context of DENV2 structural proteins, both for the WNV and DENV2 NS2B/NS3 protease, and the consequences for the production of pseudoviruses. Infectivity was determined using a WNV reporter replicon, since the efficiency of pseudovirus production is higher with this than with a similar DENV replicon [21]. However, the relative cleavage efficiency of both the WNV and DENV2 proteases was found to be similar.…”

“…1c). As the DENV2 Ca is not cleaved by the WNV NS2B/NS3 protease, we used a Ca sequence with a mutation on the cytosolic cleavage site (T101G; henceforth referred to as wild-type, WT) that is partially cleaved and restores pseudovirus production [19,21].…”