Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

International audienceThe viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. © 2017 Dubrau et al

Dom zu Fritzlar, Nordquerhaus. Fassadeninstandsetzung. Fugenarbeiten Dokumentation der Restaurierungsarbeiten am Querhaus Nord 1991

Videofilm (19 min) und BrochuereAvailable from TIB Hannover: YG3(1) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman

Polyprotein driven formation of two independent sets of complexes supporting hepatitis C virus genome replication

Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from ORF2 and a second duplicate NS polyprotein from ORF1. Using these constructs and other methods, we have assessed polyprotein requirements needed for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings; those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue; one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N) and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, a S1977P mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two inter-dependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.IMPORTANCE:Positive strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing sub-genomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behaviour of distinct NS3 helicase knock-out mutations hints that certain conformations of this enzyme might be particularly deleterious for replication

The presence of NS2 contributes to a decreased NS3/4A-kink interaction in the NS2-3/4A complex.

<p>(A) Schematic depiction of the pCITE constructs. pCITE Ubi-NS3-4A_(1–49)-TEV-GST is described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006134#ppat.1006134.g003" target="_blank">Fig 3</a>. pCITE p7-NS2-3-4A_(1–49)-TEV-GST encodes full-length NS2-3; p7 sequence was introduced upstream of NS2 (purple) to allow for correct processing of the N terminus of NS2. (B) Scheme of the TEV^pro protection assay. In the NS3/4A complex, the NS4A-kink region (kink) interacts with NS3 (red) thereby reducing TEV^pro cleavage (left panel). The presence of uncleaved NS2-3 exposes the TEV cleavage site and allows for increased TEV^pro-mediated cleavage (right panel). NS4A (blue), TEV cleavage site (grey), GST (yellow) and the NS4A-central peptide (arrow) are indicated. (C) Western blot analysis of the TEV^pro protection assay. Huh7-T7 cells were infected with MVA-T7pol vaccinia virus and incubated for 1 h at 37°C followed by co-transfection with the expression constructs indicated above, either without (left part) or with an TEV-protease expression plasmid (+HA-TEV^pro). Western blot analyses with anti-NS3, anti-GST and anti-HA are shown; for details see legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006134#ppat.1006134.g003" target="_blank">Fig 3</a>. (D) Quantification of NS4A_(1–49)-TEV-GST cleavage. Mean values and standard deviations of three independent experiments are depicted. The significance of the differences between the NS3 and the NS2-3 variant was calculated by Student’s t-test (* P = 0.013).</p

Hypothetical model of pestiviral genome replication and virion morphogenesis in presence and in absence of uncleaved NS2-3.

<p>(Left) In the pestiviral wild-type situation the NS3/4A complex is an essential component of the viral replicase while NS2-3/4A is only functional in infectious particle formation. The NS2 moiety in the NS2-3/4A complex induces the open conformation required for its packaging function. NS3 (red), NS2 (purple), uncleaved NS2-3, NS4A (blue), the NS4A central peptide (blue arrow) and the NS4A-kink region (kink). (Right) In the absence of uncleaved NS2-3 pestiviruses require gain of function mutations in NS2 and the NS3/4A complex (NS2-3-independent). The present study indicates that a conformational change at the NS3/4A-kink interface, induced by the indicated mutations, represents a molecular switch between a closed (favored during RNA replication) and an open conformation (favored during viral packaging). Individual mutations in NS2 (E440V), NS3 (V132A) and NS4A (L45A, or Y47A) which allow for NS2-3-independent virion formation are indicated.</p

Crystal structure of the CSFV NS3/4A complex.

<p>(A) Schematic diagram of the pestivirus polyprotein indicating the individual mature proteins (boxes) and proteolytic cleavage sites (arrows) (top). Scheme of the construct NS4A₃₇NS3 used for crystallization (bottom). (B) Ribbon diagram of the two molecules of CSFV NS4A₃₇NS3 in the asymmetric unit in two orientations. The two molecules within the AU of the crystals are related by a local 2-fold axis (perpendicular to the plane of the figure) around which D1 and D3 of the two helicase domains make a head-to-tail crystallographic dimer interaction. The intersection of the 2-fold axis with the paper is represented here by a central black dot (left panel). Right panel shows an orthogonal view of the left panel. One molecule is colored following the color code use in panel (A): yellow: helicase domain, red: protease domain, blue: cofactor domain, pink: GSGS linker and the other one is colored in grey. The arrows in the right panel indicate that the C-terminal end of one protomer lies in the protease active site of the other. (C) Ribbon diagram of the two molecules present in the asymmetric unit showing the protease/cofactor complex (red/blue) relative to the helicase domain (yellow). The protease domain adopts two different orientations relative to the helicase domain, giving rise to an elongated and compact conformation of the enzyme. Catalytic residues in the protease domain are depicted in green.</p