Rob W. H. Ruigrok

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R‐factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co‐ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.

Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent.

The influenza virus genome consists of eight segments of negative‐sense RNA, i.e. the viral (v) RNA forms the template for the mRNA. Each segment is encapsidated by the viral nucleoprotein to form a ribonucleoprotein (RNP) particle and each RNP carries its own polymerase complex. We studied the interaction of purified nucleoprotein with RNA in vitro, by using a variety of enzymatic and chemical probes for RNA conformation. Our results suggest that the nucleoprotein binds to the vRNA backbone without apparent sequence specificity, exposing the bases to the outside and melting all secondary structure. In this way, the viral polymerase may transcribe the RNA without the need for dissociating the nucleoprotein and without being stopped by RNA secondary structure, and the viral RNPs are ready to start transcription as soon as they enter the host cell.

Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure

Influenza virus transcription and replication is performed by ribonucleoprotein particles (RNPs). They consist of an RNA molecule covered with many copies of nucleoprotein (NP) and carry a trimeric RNA polymerase complex. RNA modification analysis and electron microscopy performed on native RNPs suggest that the polymerase forms a complex with both conserved viral RNA (vRNA) ends, whereas NP binding exposes the RNA bases to the solvent. After chemical removal of the polymerase, the bases at the vRNA extremities become reactive to modification and the vRNPs behave as structures with free ends, as judged from the observation of salt‐induced conformational changes by electron microscopy. The vRNA appears to be completely single‐stranded in polymerase‐free RNPs despite a partial, inverted complementarity of the vRNA ends. The absence of a stable double‐stranded panhandle structure in polymerase‐free RNPs has important implications for the mechanism of viral transcription and the switch from transcription to replication.

Rabies virus glycoprotein is a trimer.

Abstract The oligomerization state of the rabies virus envelope glycoprotein (G protein) was determined using electron microscopy and sedimentation analysis of detergent solubilized G. Most of the detergents used in this study solubilized G in a 4 S monomeric form. However, when CHAPS was used, G had a sedimentation coefficient of 9 S. This high sedimentation coefficient allowed its further separation from M1 and M2. Using electron microscopy of negatively stained samples, we studied the morphology of G on virus and after detergent extraction. End-on views of G on virus clearly showed triangles consisting of three dots indicating the trimeric nature of native G. End-on views of CHAPS-isolated G showed very similar triangles confirming that, using this detergent, G was solubilized in its native trimeric structure. Electron microscopy also showed that G had a “head” and a “stalk” and provided the basis for a low-resolution model of the glycoprotein structure.

Structural insight into cap-snatching and RNA synthesis by influenza polymerase.

Influenza virus polymerase uses a capped primer, derived by ‘cap-snatching’ from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.

The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.

Tetrameric coiled coil domain of Sendai virus phosphoprotein

The high resolution X-ray structure of the Sendai virus oligomerization domain reveals a homotetrameric coiled coil structure with many details that are different from classic coiled coils with canonical hydrophobic heptad repeats. Alternatives to the classic knobs-into-holes packing lead to differences in supercoil pitch and diameter that allow water molecules inside the core. This open and more hydrophilic structure does not seem to be destabilized by mutations that would be expected to disrupt classic coiled coils.

Intrinsic disorder in measles virus nucleocapsids

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (NTAIL) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of NTAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (NCORE) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of NTAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that NTAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with NCORE. We present a model in which the first 50 disordered amino acids of NTAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive NCORE helical turns. The model provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

Low-pH induced conformational changes in viral fusion proteins: implications for the fusion mechanism

Introduction. Entry of enveloped viruses into host cells requires binding of the virus to one or more receptors present at the host cell surface followed by fusion of the viral envelope with a cellular membrane. After binding, viruses such as paramyxoviruses, retroviruses and herpesviruses are thought to fuse directly with the plasma membrane. For other viruses, including the alpha-, rhabdo- and ortho-myxoviruses, binding does not directly lead to fusion. Instead, the bound virus particles are first internalized and then, at the low-pH within this compartment, fuse with the endosomal membrane. In this review, we will focus on this latter class of viruses. For alpha-, rhabdo- and orthomyxoviruses, a glycoprotein is responsible for both virus attachment and fusion. In the acidic environment of the endosome, the ectodomain portion of the glycoprotein undergoes a major structural rearrangement to generate a fusion-competent state.

Quantitative conformational analysis of partially folded proteins from residual dipolar couplings: application to the molecular recognition element of Sendai virus nucleoprotein.

A significant fraction of proteins coded in the human proteome do not fold into stable three-dimensional structures but are either partially or completely unfolded. A key feature of this family of proteins is their proposed capacity to undergo a disorder-to-order transition upon interaction with a physiological partner. The mechanisms governing protein folding upon interaction, in particular the extent to which recognition elements are preconfigured prior to formation of molecular complexes, can prove difficult to resolve in highly flexible systems. Here, we develop a conformational model of this type of protein, using an explicit description of the unfolded state, specifically modified to allow for the presence of transient secondary structure, and combining this with extensive measurement of residual dipolar couplings throughout the chain. This combination of techniques allows us to quantitatively analyze the level and nature of helical sampling present in the interaction site of the partially folded C-terminal domain of Sendai virus nucleoprotein (N(TAIL)). Rather than fraying randomly, the molecular recognition element of N(TAIL) preferentially populates three specific overlapping helical conformers, each stabilized by an N-capping interaction. The unfolded strands adjacent to the helix are thereby projected in the direction of the partner protein, identifying a mechanism by which they could achieve nonspecific encounter interactions prior to binding. This study provides experimental evidence for the molecular basis of helix formation in partially folded peptide chains, carrying clear implications for understanding early steps of protein folding.