Biology subjects

Carillo, K. J. D.

Publications and source records attributed to Carillo, K. J. D..

3 recordsLinked to original sources

bioRxiv2026

Structure and functional analyses of vaccinia virus J5 protein reveal distinct determinants for entry-fusion complex assembly and activation

Vaccinia virus enters host cells through a multi-component entry fusion complex (EFC) that is structurally distinct from canonical viral fusion systems. Understanding how vaccinia virus EFC mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we report the solution NMR structure of a truncated ectodomain of vaccinia J5 protein, residues 2-68. Using recombinant vaccinia viruses expressing J5 mutants, we analyzed substitutions in conserved and surface-exposed residues, as well as chimeric constructs between vaccinia J5 and its entomopoxvirus ortholog AMV232. Functional analyses revealed that the conserved P38YYCWY43 motif is dispensable for EFC assembly but required for membrane fusion activity whereas the flexible region spanning residues 90-110 mediates interactions required for stable incorporation of J5 into the EFC. ImportanceVaccinia virus enters host cells through membrane fusion mediated by a unique multi-component entry fusion complex (EFC) that is distinct from classical viral fusion proteins. Although J5 has been identified as a central component of the pre-fusion EFC, the structural regions of J5 required for membrane fusion remain unclear. Here, we determined the solution NMR structure of the J5 ectodomain and identified two regions, the conserved P38YYCWY43 motif and residues 90-110, as key determinants of EFC function during vaccinia virus entry.

microbiology↗

bioRxiv2024

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Nervous necrosis virus (NNV), a non-enveloped betanodavirus, causes neuropathies and retinopathies in farmed fish, damaging aquaculture worldwide. NNV has 60 conspicuous surface protrusions comprising the protrusion domain (P-domain) of its capsid protein. Although NNV protrusions play critical roles in infectivity, the underlying dynamics remain unclear. Our cryogenic electron microscopy (cryo-EM)-derived structures of Dragon grouper (Epinephelus lanceolatus) NNV reveal that the protrusions undergo low-pH-induced compaction and movement. We show that the P-domain is monomeric in solution at a pH germane to infection (7.0). Moreover, nuclear magnetic resonance (NMR) structures reveal a peptide (amino acids 311-330) that adopts a flexible loop to form an open pocket. NMR spectral analysis at pH 5.0 aided by molecular dynamics (MD) simulations show that this loop switches to a {beta}-strand under acidic conditions, eliciting pocket closure and P-domain trimerization, highlighting a unique pH-sensing feature. Our docking analysis revealed the N-terminal moiety of sialic acid inserted into and interacting with conserved residues in the pocket. Additionally, a low-pH-induced conformational change in the linker region via peptide bond isomerization conferred malleability on the protrusions. Our work uncovers the protrusion dynamics of a betanodavirus governing its infectivity through a pH-dependent conformational switching mechanism, providing insights into complex virus-host interactions.

biochemistry↗

bioRxiv2023

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex

Virus-mediated membrane fusion involves conformational changes of the viral fusion protein to fuse the opposing viral and host lipid bilayers. Unlike all other known viruses that contain a single fusion protein, poxviruses harbor a multimeric protein complex of 11 subunits, termed the entry fusion complex (EFC), to mediate fusion with host membranes. Yet, how the poxviral EFC mediates membrane fusion remains enigmatic. To establish the mechanism of EFC-triggered membrane fusion, we are deciphering the structure and function of individual EFC components. Here, we determined the crystal structure of the H2 ectodomain by X-ray diffraction, revealing a folded conformation comprising a central five-stranded {beta}-sheet and three cladding -helices. We reconstructed the full-length H2 by in silico prediction, revealing that the N-terminal region (aa 51-90) of H2 protein may fold as a long helix connecting the ectodomain and transmembrane region. Using alanine-mutagenesis screening in a transient complementation system, coimmunoprecipitation, isothermal titration calorimetry and MV-triggered membrane fusion assays, we concluded that the surface of the ectodomain of H2 protein, including two loop regions, 170LGYSG174 and 125RRGTGDAW132, constitutes a broad A28-binding region. Moreover, although not involved in A28 binding, the N-terminal helical region approximal to the transmembrane part, encompassing 64RIK66, 72W, and 83ESDRGR88, is also crucial for viral EFC formation and MV infectivity.

microbiology↗