Gerd Wengler

The Fusion Glycoprotein Shell of Semliki Forest Virus: An Icosahedral Assembly Primed for Fusogenic Activation at Endosomal pH

Semliki Forest virus (SFV) has been extensively studied as a model for analyzing entry of enveloped viruses into target cells. Here we describe the trace of the polypeptide chain of the SFV fusion glycoprotein, E1, derived from an electron density map at 3.5 A resolution and describe its interactions at the surface of the virus. E1 is unexpectedly similar to the flavivirus envelope protein, with three structural domains disposed in the same primary sequence arrangement. These results introduce a new class of membrane fusion proteins which display lateral interactions to induce the necessary curvature and direct budding of closed particles. The resulting surface protein lattice is primed to cause membrane fusion when exposed to the acidic environment of the endosome.

The carboxy-terminal part of the NS 3 protein of the West Nile Flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase

Recently it has been reported that a membrane fraction can be isolated from West Nile virus-infected BHK cells which contains the viral nonstructural (NS) proteins as major constituents (Wengler et al., 1990). In this report we show that treatment of these membranes with subtilisin releases the carboxy-terminal segment of the NS 3 protein as a soluble protein of about 50 kDa apparent molecular weight. This molecule, which is called the p50-S protein, can be purified by standard chromatographic procedures. The p50-S protein binds to poly(A) and apparently represents a nucleoside triphosphatase which is stimulated in the presence of ssRNA molecules. The data represent experimental support for the predicted role of this segment of the NS 3 protein as an RNA helicase. Some properties of the p50-S protein are described and a possible function of this protein segment during RNA synthesis is discussed.

Studies on virus-specific nucleic acids synthesized in vertebrate and mosquito cells infected with flaviviruses

Abstract Virus-specific RNA molecules synthesized in BHK 21 vertebrate cells and in Aedes albopictus mosquito cells infected with the flaviviruses Uganda S (US) or West Nile (WN) have been characterized. A single-stranded (ss) RNA of plus polarity sedimenting at about 42 S was present in the virus particles. 42 S plus strand RNA was also the predominant species of virus-specific ss RNA accumulating in infected cells of both vertebrate and insect origin. No similarity was detected between the large oligonucleotides generated by ribonuclease T 1 from WN virus and US virus-specific 42 S plus strand RNA, respectively. No poly(A) sequences are present in either the US virus or the WN virus-specific 42 S plus strand RNA molecules synthesized in BHK cells. The 42 S plus strand RNA molecules present in WN virus-infected BHK cells do contain, however, a “cap” structure m 7 GpppAmpN 1 , and in some of these molecules, a further methyl group is introduced, giving rise to the “cap” structure m 7 GpppAmpN 1 mpN 2 . These “caps” are also present on the 42 S RNA of WN virus particles synthesized in BHK cells. In addition to the 42 S RNA, virus-specific ss RNA of low molecular weight (LMW-RNA) was detected in all virus-cell systems analyzed. A single species of LMW-RNA of 5 × 10 4 daltons apparent molecular weight was present in US virus-infected vertebrate and insect cells. Two LMW-RNA species of about 6.5 × 10 4 daltons (WM LMW-1 RNA) and 4.2 × 10 4 daltons (WN LMW-2 RNA) molecular weight, respectively, were isolated from WN virus-infected BHK cells. Only the larger of these was detected in WN virus-infected insect cells. The LMW-1 RNA present in WN virus-infected BHK cells has been characterized in somewhat more detail. It contains virus-specific RNA sequences of plus strand polarity, is not “capped”, and does not contain a poly(A) sequence. None of the single-stranded, virus-specific RNA molecules synthesized in either vertebrate or mosquito cells bound to oligo(dT)-cellulose. Only a single species of virus-specific RNA containing minus strand sequences was detected in WN virus-infected BHK cells. This RNA was of genome size and was present as part of a double-stranded RNA complex containing 42 S RNA of both plus and minus polarity.

Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus west nile virus and of the genome sequence for these proteins

Cell-associated flaviviruses contain the two membrane proteins V3 and NV2 besides the viral core protein V2 whereas extracellular viruses do contain V2 protein and the two membrane proteins V3 and V1. Since the V1 protein could not be detected in infected cells it has been suggested that V1 is generated from NV2 by proteolytic cleavage during the release of virus from cells (D. Shapiro, W. E. Brandt, and P. K. Russell (1972), Virology 50, 906-911). We have isolated the viral structural proteins V1, V2, and NV2 from the flavivirus West Nile virus and determined their amino-terminal amino acid sequences and amino acid sequences of peptides derived from these proteins. We have also transcribed parts of the viral genome into cDNA and cloned and sequenced this cDNA. The analyses of the protein structure of V1, V2, and NV2 together with the determination of the amino-terminal sequence of V3 (data not shown) have allowed us to identify the nucleotide region coding for the structural proteins V2, NV2, and V1. The primary structure of this nucleotide sequence is presented in this report. The data show that the amino terminus of the viral core protein V2 is followed by the amino termini of the proteins NV2, V1, and V3, respectively. These data for the first time identify the exact order of all structural proteins of a flavivirus identified so far. Our data strongly support the above-mentioned hypothesis that V1 is derived from NV2 by proteolytic cleavage and furthermore indicate that V1 represents the nonglycosylated carboxy-terminal part of NV2 which contains those sequences which anchor NV2 in the viral membrane. A working hypothesis is presented in which two species of cellular enzymes, signalase(s) removing signal sequences and enzymes involved in cleaving polyproteins after a pair of basic amino acids, do generate the proteins V2, NV2, and V1 from the growing peptide chain synthesized during translation of the 42 S genome RNA which functions as mRNA for these proteins.

Analyses of the terminal sequences of west nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre-M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues 1-105, 124-290, and 291-787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells.

In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids.

A virus-encoded protease that cleaves after multiple basic amino acid residues has been implicated in the processing of the flavivirus polyprotein. Recently, a computer search of amino acid residues which might form the active site of a protease led to the suggestion that the amino-terminal segment of the NS3 protein represents a serine protease. To examine this possibility we constructed an mRNA which encodes a polyprotein with an amino-terminal signal sequence derived from the influenza virus haemagglutinin, followed by a segment of the West Nile flavivirus polyprotein which includes the non-structural (NS) proteins NS2A, NS2B and the amino-terminal part of the NS3 protein. This polyprotein contains two sequences, located at the termini of the NS2B protein, which are cleaved by the viral protease that cleaves after multiple basic residues in the authentic polyprotein. The proteins that are generated by this mRNA during in vitro translation in the presence of rough endoplasmic reticulum membranes indicate that these two proteolytic cleavages occur in vitro. In vitro translation of polyproteins shortened at the carboxy terminus shows that a polyprotein which does not contain the complete set of proposed catalytic residues present in the NS3 protein segment accumulates as a membrane-associated molecule without proteolytic processing. Similarly, substitution of residue histidine 51 of the NS3 polyprotein segment, which is predicted to be part of the protease catalytic centre, with an alanine residue, blocks the processing of the polyprotein in vitro.

Terminal sequences of the genome and replicatioe-form RNA of the flavivirus west nile virus: absence of poly(A) and possible role in RNA replication

Abstract The structures of the infectious 42 S genome RNA of the flavivirus West Nile (WN) virus and of the replicative-form (RF) RNA containing 42 S RNA of positive and negative polarity have been investigated. The RF RNA has been labeled in vitro at the 3′ and 5′ termini and the terminal sequences have been determined by the mobility shift method. The results obtained indicate that both RNA molecules are exact complements of each other and that the 3′ terminus of the 42 S plus-strand RNA component of the RF RNA does not contain a poly(A) sequence but terminates with a heteropolymeric AACACAGGAUCU OH sequence. The 3′ terminus of the 42 S minus-strand RNA has the sequence CUCACACAGGCGAACUACU OH . Comparison of these sequences shows that both molecules contain the 3′-terminal dinucleotide CUOH and the heptanucleotide ACACAGG which is separated from the 3′-terminal dinucleotide by two and seven nucleotides in 42 S plus- and minus-strand RNA, respectively. The 42 S viral genome RNA also does not contain a 3′-terminal poly(A) sequence but terminates with the 3′-terminal sequence identified in the 42 S plus-strand RNA of the RF. Analysis of the nucleotides adjacent to the cap at the 5′ terminus of the viral genome RNA together with the 3′-terminal sequence analysis indicates that the nucleotide sequence of the viral genome RNA is identical to that of the 42 S plus-strand RNA component of the virus-specific RF RNA.

Structure of Semliki Forest virus core protein.

Alphaviruses are enveloped, insect‐borne viruses, which contain a positive‐sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin‐like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy‐terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 Å and 3.3 Å resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail‐to‐tail dimer contact, monomers B and C make a head‐to‐head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head‐to‐tail contacts found in the pentameric and hexameric associations within the virion utilize the same monomer surface regions as found in the crystalline dimer interfaces. Proteins 27:345–359, 1997.

Analysis of disulfides present in the membrane proteins of the West Nile flavivirus

Recently the primary structure of the structural proteins of the flaviviruses West Nile (WN) virus (Castle et al., 1985; Wengler et al., 1985) and yellow fever (YF) virus (Rice et al., 1985) have been determined. As a first step in a further characterization of the organization of the structural proteins we have now studied the disulfide bridges present in the WN virus membrane proteins. All three membrane proteins, pre M, M, and E, were analyzed. The results obtained can be summarized as follows: The pre M proteins of both WN and YF virus each contain 6 cysteine residues and the position of all of these residues is strictly conserved between both viruses. The M proteins of both viruses do not contain cysteine residues. The E proteins of these viruses contain 12 cysteines and the position of all of these residues is strictly conserved between both viruses. All cysteine residues of the WN virus-derived membrane proteins are present as intramolecular disulfides. The six disulfide bridges generated from the 12 cysteine residues in the WN virus-derived E protein have been identified as follows: Cys 1-Cys 2; Cys 3-Cys 8; Cys 4-Cys 6; Cys 5-Cys 7; Cys 9-Cys 10; Cys 11-Cys 12. The analyses of the amino acid sequence conservation between the E proteins of YF and WN virus and the characterization of the disulfides have been used to develop a description of the E protein in which the molecule is assumed to be composed of the segments R1, L1, R2, L2, and R3 followed by a membrane anchor region at the carboxy-terminal region of the molecule. Computer analyses of the hydrophilicity and of the secondary structure indicate that the R1 region might contain a cluster of viral epitopes.

Identification of a sequence element in the alphavirus core protein which mediates interaction of cores with ribosomes and the disassembly of cores

Early in infection core protein is transferred from alphavirus cores to ribosomes (Wengler and Wengler, 1984, Virology 134, 435-442) and it has been suggested that ribosome binding is a property of alphavirus core protein which is involved in core disassembly. Here we describe in vitro analyses of this transfer. Sindbis virus cores, incubated with ribosomes either in a reticulocyte lysate or in buffer, are disassembled with a concomitant transfer of core protein to the large ribosomal subunit. Preincubation of ribosomes with core protein blocks disassembly. Limited proteolysis of Sindbis virus core releases the carboxy-terminal core protein domain as a soluble fragment (Strong and Harrison, 1990, J. Virol. 64, 3992-3994). Trypsin- or proteinase Lys-C-released fragments contain the amino-terminal residue met (106) or gln (94), respectively. The fragment generated by proteinase Lys-C binds to ribosomes and interferes with core disassembly whereas the slightly shorter tryptic fragment has none of these activities. These and further analyses indicate that a conserved sequence element which surrounds amino acid met (106) of SIN CP, the so-called RBSc element, leads to binding of core protein to ribosomes and thereby to core disassembly. Implications of the experiments for regulation of assembly of alphavirus cores and for the core protein-induced resistance to viral multiplication observed in plant virus systems are discussed.