Disulfide-bond formation by a single cysteine mutation in adenovirus protein VI impairs capsid release and membrane lysis

AbstractThe internal capsid protein VI mediates adenovirus (AdV) endosome penetration during cell entry. Essential to this process is the release of protein VI from the AdV capsid and subsequent membrane targeting and insertion by the liberated VI molecules within the endocytic vesicle. In this study, we describe a human AdV (HAdV) substitution mutant (AdV VI-G48C) within the critical N-terminal amphipathic α-helical domain of protein VI. The VI-G48C virus displays altered capsid stability that impacts protein VI release, membrane disruption and virus infectivity. This is due in part to aberrant disulfide-bonding of protein VI molecules within the AdV particle. Our results provide insight into the structural organization of protein VI in the virus particle, as well as highlight the role of protein VI in cell entry

Functional Genetic and Biophysical Analyses of Membrane Disruption by Human Adenovirus▿

The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions

The cleaved N-terminus of pVI binds peripentonal hexons in mature adenovirus

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule

The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

<div><p>Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using <i>in vitro</i> biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.</p></div

Mutation of the VP30 NP-binding site alters replicase activity.

<p>A) Mini-replicon activities are presented as a percentage of the firefly luciferase activity observed with wild-type VP30. Dissociation constants observed in ITC experiments are listed for each mutant. B) Western blot for NP, VP35 and VP30 of cleared lysates from cells transfected with the mini-replicon system, confirming similar levels of expression compared for VP30 mutants to wild-type.</p

Conservation of NP and VP30 interactions.

<p>A) Sequences of filovirus NP 600–617 (EBOV numbering) were aligned with Clustal Omega [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.ref019" target="_blank">19</a>]. Residues identical to EBOV are colored green and similar residues are in yellow. B) Sequences of filovirus VP30 C-terminal region were aligned with Clustal Omega [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.ref019" target="_blank">19</a>]. Those amino acids of the VP30 CTD that are visible in the EBOV VP30-NP complex crystal structure are colored blue. VP30 amino acids within 5 Å of EBOV NP 602–614 are indicated in purple.</p

Structural Basis of Pan-Ebolavirus Neutralization by a Human Antibody against a Conserved, yet Cryptic Epitope

There are five different members of the Ebolavirus genus. Provision of vaccines and treatments able to protect against any of the five ebolaviruses is an important goal of public health. Antibodies are a desired result of vaccines and can be delivered directly as therapeutics. Most antibodies, however, are effective against only one or two, not all, of these pathogens. Only one human antibody has been thus far described to neutralize all five ebolaviruses, antibody ADI-15878. Here we describe the molecular structure of ADI-15878 bound to the relevant target proteins of Ebola virus and Bundibugyo virus. We explain how it achieves its rare breadth of activity and propose strategies to design improved vaccines capable of eliciting more antibodies like ADI-15878.Only one naturally occurring human antibody has been described thus far that is capable of potently neutralizing all five ebolaviruses. Here we present two crystal structures of this rare, pan-ebolavirus neutralizing human antibody in complex with Ebola virus and Bundibugyo virus glycoproteins (GPs), respectively. The structures delineate the key protein and glycan contacts for binding that are conserved across the ebolaviruses, explain the antibody’s unique broad specificity and neutralization activity, and reveal the likely mechanism behind a known escape mutation in the fusion loop region of GP2. We found that the epitope of this antibody, ADI-15878, extends along the hydrophobic paddle of the fusion loop and then dips down into a highly conserved pocket beneath the N-terminal tail of GP2, a mode of recognition unlike any other antibody elicited against Ebola virus, and likely critical for its broad activity. The fold of Bundibugyo virus glycoprotein, not previously visualized, is similar to the fold of Ebola virus GP, and ADI-15878 binds to each virus’s GP with a similar strategy and angle of attack. These findings will be useful in deployment of this antibody as a broad-spectrum therapeutic and in the design of immunogens that elicit the desired broadly neutralizing immune response against all members of the ebolavirus genus and filovirus family

Analysis of RNA levels shows the VP30-NP interaction to be a general modulator of RNA synthesis activity.

<p>RNA from mini-replicon transfected cells was reverse transcribed using primers specific for vRNA (blue), cRNA (red) or mRNA (green). Quantitative PCR was performed with primers amplifying the firefly luciferase ORF common to all RNA types. VP30 mutants are indicated and are arranged from high affinity (left) to low affinity (right) binders. Wild-type (WT) and negative controls (-L, -3E5E-ffLuc, -DNA) are presented for comparison. Data are normalized to the wild-type control samples.</p

EBOV VP30 mutations to the NP-binding site alter the affinity of the interaction.

<p>EBOV VP30 mutations to the NP-binding site alter the affinity of the interaction.</p

The gradient of VP30-NP interaction affinities shows two phases of RNA synthesis activity.

<p>Increasing affinity (decreasing K_D) of the VP30-NP interaction (blue) from wild-type (WT), as assessed by multiple assays (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.t002" target="_blank">Table 2</a>, Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g004" target="_blank">4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g007" target="_blank">7</a>), represses viral RNA synthesis activity (pink) (Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g008" target="_blank">8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g009" target="_blank">9</a>) while mildly decreasing affinity activates viral RNA synthesis. A transition occurs beyond the affinity of the VP30 D202R mutant (dashed line) such that further reductions in affinity result in diminished RNA synthesis activity reflecting the essential nature of the VP30-NP interaction to the RNA synthesis complex.</p