Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins

Studies on three-way DNA junctions related to the development of a novel method for the detection of genetic polymorphisms

Mutations in tlie genetic material of living organisms can have major effects on the survival of the individual. Genetic variations influence both the virulence and treatment-susceptibility of micro-organisms and affect the host-susceptibility for pathogenic micro-organisms. In addition, many common genetic disorders arise from defects in multiple alleles. This has important implications for health diagnostics as most available methodologies for genetic screening are not suitable to detect these. We have therefore developed a novel nucleic acid screening method, named signal mediated amplification of RNA technology (SMART), for detection of genetic polymorphisms. The technology uses two nucleic acid probes that anneal to a specific target sequence and to each other, resulting in the formation of a three- way DNA junction (TWJ). One probe (extension probe) can be extended against the remaining probe (template probe) by a DNA polymerase. This activity yields a functional T7 RNA polymerase promoter, and addition of T7 RNA polymerase results in RNA production. The RNA produced functions as a signal for the presence of a specific target sequence. These studies have focussed on optimising the junction design in SMART to allow discrimination between targets containing only small genetic variations. The results indicate that a number of factors can enhance the specificity of SMART: 1) incorporation of PNA (peptide nucleic acids) in the target complementary regions of the two probes, 2) shortening the size of the target containing duplex arms, 3) shortening the complementary sequence shared by the two probes, 4) elevating the reaction temperature. A further DNA-analogue (Locked Nucleic Acids, LNA) was also assessed but found to be less sensitive for mutation detection. The optimised assay is able to discriminate between the wild-type and single- and three-base variants of the cystic fibrosis transmembrane conductance regulator and Factor V genes, using either synthetic or PGR amplified genomic material. Additional studies also revealed that the structure of the TWJ may affect the RNA yield. In the presence of divalent cations, three-way junctions adopt two different coaxially stacked confonnations. The preferred conformer depends on the sequence around the branch point and this affects the efficiency of the assay. We have used a gel-electrophoresis method to probe the structures adapted by different junctions. The major factor influencing the conformer distribution is the arrangement of purines and pyrimidines around the junction, although some junctions appeared to adopt unusual conformations. Together these findings have improved the understanding of TWJ conformation and allow for more accurate structure prediction based only on the DNA sequence. Finally, a novel cleavage activity of restriction endonucleases was discovered and characterised. The activity was observed only for DNA constructs containing a non-DNA linker (e.g. hexaethylene glycol, abasic site) and involved cleavage of the phosphate located directly 5' of the linker. The cleavage was further found to be independent of the presence of canonical substrate and appeared to be modulated by methyltransferase treatment.</p

A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1

International audienceThe structure-specific DNA-binding protein HMGB1, which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys-2 and Lys-11 in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of double mutation on four-way junction binding, although binding to minicircles is reduced ~ 15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys-2 or Lys-11 abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys-2 and Lys-11 are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates

Crystal structure of a novel conformational state of the flavivirus NS3 protein: Implications for polyprotein processing and viral replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed. Copyright © 2009, American Society for Microbiology. All Rights Reserved

Structure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein▿

Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae

Semi-automated microseeding of nanolitre crystallization experiments

A procedure for microseeding into nanolitre crystallization drops is described with selected successful examples