The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal globular (head) domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar ‘difficult’ viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions

We have recently developed a mouse monoclonal antibody (12–10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12–10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the “hot spot” in the 12–10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12–10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the “hot spot” in the paratope.

Short-stalk isoforms of CADM1 and CADM2 trigger neuropathogenic measles virus-mediated membrane fusion by interacting with the viral hemagglutinin

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae , usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We have recently shown that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors. Importance Measles, an acute febrile illness with skin rash, is still an important cause of childhood morbidity and mortality worldwide. Measles virus (MeV), the causative agent of measles, may also cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. The disease is fatal, and no effective therapy is available. Recently, we have reported that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV cell-to-cell spread in neurons. These molecules interact in cis with the MeV attachment protein on the same cell membrane, triggering the fusion protein and causing membrane fusion. CADM1 and CADM2 are known to exist in multiple splice isoforms. In this study, we report that their short-stalk isoforms can induce membrane fusion by interacting in cis with the viral attachment protein independently of its receptor-binding head domain. This finding may have important implications for cis -acting fusion triggering by host factors.

The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal head domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar 'difficult' viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Design of the Recombinant Influenza Neuraminidase Antigen is Crucial for its Biochemical Properties and Protective Efficacy

Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving the suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced using the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and were compared for enzymatic property, antigenicity, stability, and protection in mice. We found that distinct enzymatic properties are associated with rNAs containing the NA head-domain versus the full-ectodomain, formation of higher order rNA oligomers is tetramerization domain-dependent, whereas protective efficacy is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, a rNA possessing the full-ectodomain and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided much better protection than a rNA with ∼10-fold more enzymatically active molecules that is comprised of the head-domain and the same tetramerization motif. In contrast, these two rNA designs provided comparable protection when the tetramerization motif from the tetrabrachion protein was used instead. These findings demonstrate that individual rNAs should be thoroughly evaluated for vaccine development, as the heterologous domain combination can result in rNAs with similar key attributes but vastly differ in protection. IMPORTANCE For several decades it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve the efficacy. However, some key questions for manufacturing stable and immunogenic rNA remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunogenic and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.

Robust maintenance of cell surface tension in mitosis by RhoA-driven myosin II mechanoresponse

As cells enter mitosis, cell cortex contraction generates surface tension to establish a geometry feasible for division in a physically confined environment. Cell surface tension rises in prophase and continues to stay constant during metaphase to support mitosis. How the cell surface tension is maintained throughout mitosis is not well explored. We show that the cell surface tension is actively maintained by a mechanosensitive RhoA pathway at the cell cortex during mitosis. Mechanical activation of RhoA leads to non-muscle myosin IIB (NMIIB) stabilization and mechanosensitive accumulation at the cell cortex via Rho kinase (ROCK) regulation of the NMIIB head domain. Interestingly, when the NMIIB tail domain regulation is perturbed, the NMIIB has reduced ability to generate tension but could still support mitotic cells to withstand compressive stress by undergoing mechanosensitive accumulation at the cell cortex. Thus, mechanical RhoA activation drives NMIIB mechanoresponse via its head domain regulation to maintain cell surface tension during mitosis.

Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

Cyclin D3 regulates the G1/S transition and is frequently overexpressed in several cancer types including breast cancer, where it promotes tumor progression. Here, we show that a cytoskeletal protein keratin 19 (K19) physically interacts with a serine/threonine kinase GSK3β and prevents GSK3β-dependent degradation of cyclin D3. The absence of K19 allowed active GSK3β to accumulate in the nucleus and degrade cyclin D3. Specifically, the head domain of K19 was required to sustain inhibitory phosphorylation of GSK3β Ser9, prevent nuclear accumulation of GSK3β, and maintain cyclin D3 levels and cell proliferation. K19 was found to interact with GSK3β and K19-GSK3β interaction was mapped out to require Ser10 and Ser35 residues on the head domain of K19. Unlike wildtype K19, S10A and S35A mutants failed to maintain total and nuclear cyclin D3 levels and induce cell proliferation. Finally, we show that the K19-GSK3β-cyclin D3 pathway affected sensitivity of cells towards inhibitors to cyclin dependent kinase 4 and 6 (CDK4/6). Overall, these findings establish a role for K19 in the regulation of GSK3β-cyclin D3 pathway and demonstrate a potential strategy for overcoming resistance to CDK4/6 inhibitors.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Hybrid QM/MM Simulations Confirm Zn(II) Coordination Sphere That Includes Four Cysteines from the P2 × 4R Head Domain

To ascertain the role of Zn(II) as an allosteric modulator on P2X4R, QM/MM molecular dynamic simulations were performed on the WT and two P2X4R mutants suggested by previous electrophysiological data to affect Zn(II) binding. The Gibbs free energy for the reduction of the putative P2X4R Zn(II) binding site by glutathione was estimated at −22 kcal/mol. Simulations of the WT P2X4R head domain revealed a flexible coordination sphere dominated by an octahedral geometry encompassing C126, N127, C132, C149, C159 and a water molecule. The C132A mutation disrupted the metal binding site, leading to a coordination sphere with a majority of water ligands, and a displacement of the metal ion towards the solvent. The C132A/C159A mutant exhibited a tendency towards WT-like stability by incorporating the R148 backbone to the coordination sphere. Thus, the computational findings agree with previous experimental data showing Zn(II) modulation for the WT and C132A/C159A variants, but not for the C132A mutant. The results provide molecular insights into the nature of the Zn(II) modulation in P2X4R, and the effect of the C132A and C132A/C159A mutations, accounting for an elusive modulation mechanism possibly occurring in other extracellular or membrane protein.