Zika virus capsid anchor forms cytotoxic amyloid-like fibrils.

Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed the aggregation behavior of CA peptide using dye-binding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM, AFM) and Far-UV CD spectroscopy revealed characteristic amyloid-like fibrils rich in ß-sheet secondary structure. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection.

DENV2 Pseudoviral Particles with Unprocessed Capsid Protein Are Assembled and Infectious.

Proteolytic processing of flavivirus polyprotein is a uniquely controlled process. To date, the sequential cleavage of the capsid anchor sequence at the junction of C-PrM has been considered essential for the production of flaviviruses. In this study, we used two experimental approaches to show the effect of unprocessed capsid on the production and infectivity of dengue virus 2 (DENV2) pseudoviral particles. The results showed that (1) both mature and unprocessed capsids of DENV2 were equally efficient in the viral RNA packaging and also in the assembly of infective particles; (2) DENV2 variants, in which the viral and host mediated cleavage of Ca peptide were independent, produced significantly higher levels of infective particles. Overall, this study demonstrated that unlike other flaviviruses, DENV2 capsid does not require a cleavable Ca sequence, and the sequential cleavage is not an obligatory requirement for the morphogenesis of infective pseudoviral particles.

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

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

Role of Capsid Anchor in the Morphogenesis of Zika Virus.

The flavivirus capsid protein (C) is separated from the downstream premembrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the endoplasmic reticulum (ER). To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of Zika virus was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression in cis with respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of heterologous, but not homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.IMPORTANCE The capsid anchor (Ca) is a single-pass transmembrane domain at the C terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further examine the role of Ca in Zika virus life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.