Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Molecular characterization of guinea pig-adapted variants of Ebola virus.

Serial passage of initially nonlethal Ebola virus (EBOV) in outbred guinea pigs resulted in the selection of variants with high pathogenicity. Nucleotide sequence analysis of the complete genome of the guinea pig-adapted variant 8mc revealed that it differed from wild-type virus by eight mutations. No mutations were identified in nontranscribed regions, including leader, trailer, and intragenic sequences. Among noncoding regions the only base change was found in the VP30 gene. Two silent base changes were found in the open reading frame (ORF) encoding NP protein. Nucleotide changes resulting in single-amino-acid exchanges were identified in both NP and L genes. Three other mutations found in VP24 caused amino acid substitutions, which are responsible for larger structural changes of this protein, as indicated by an alteration in electrophoretic mobility. A highly pathogenic EBOV variant K5 from another passaging series showed an amino acid substitution at nearly the same location in the VP24 gene, suggesting the importance of this protein in the adaptation process. In addition, sequence variability of the GP gene was found when plaque-purified clones of EBOV-8mc were analyzed. Three of five viral clones showed insertion of one uridine residue at the GP gene-editing site, which led to a significant change in the expression of virus glycoproteins. This observation suggests that the editing site is a hot spot for insertion and deletion of nucleotides, not only at the level of transcription but also of genome replication. Irrespective of the number of uridine residues at the editing site, all plaque-purified clones of EBOV variant 8mc resembled each other in their pathogenicity for guinea pigs, indicating either the absence or only supportive role of mutations in the GP gene on the adaptation process.

Proteolytic processing of Marburg virus glycoprotein.

Processing of the transmembrane glycoprotein (GP) of Marburg virus involved the conversion of an endo H-sensitive, ER-specific form into an endo H-resistant, Golgi-specific precursor that was cleaved into GP(1) and GP(2). Cleavage was mediated by furin or another subtilisin-like endoprotease with similar substrate specificity as indicated by mutational analysis of the cleavage site and inhibition using peptidyl chloromethylketones. Mature GP consisted of disulfide-linked GP(1) and GP(2) subunits.

Delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sGP of Ebola virus.

In the present study we have investigated processing and maturation of the nonstructural small glycoprotein (sGP) of Ebola virus. When sGP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments using SDS-PAGE under reducing conditions, the mature form and two different precursors have been identified. First, the endoplasmic reticulum form sGP(er), full-length sGP with oligomannosidic N-glycans, was detected, sGP(er) was then replaced by the Golgi-specific precursor pre-sGP, full-length sGP containing complex N-glycans. This precursor was finally converted by proteolysis into mature sGP and a smaller cleavage fragment, Delta-peptide. Studies employing site-directed mutagenesis revealed that sGP was cleaved at a multibasic amino acid motif at positions 321 to 324 of the open reading frame. Cleavage was blocked by RVKR-chloromethyl ketone. Uncleaved pre-sGP forms a disulfide-linked homodimer and is secreted into the culture medium in the presence of the inhibitor as efficiently as proteolytically processed sGP. In vitro treatment of pre-sGP by purified recombinant furin resulted in efficient cleavage, confirming the importance of this proprotein convertase for the processing and maturation of sGP. Delta-peptide is also secreted into the culture medium and therefore represents a novel nonstructural expression product of the GP gene of Ebola virus. Both cleavage fragments contain sialic acid, but only Delta-peptide is highly O-glycosylated.

The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

Characterization of the L gene and 5' trailer region of Ebola virus.

The nucleotide sequences of the L gene and 5' trailer region of Ebola virus strain Mayinga (subtype Zaire) have been determined, thus completing the sequence of the Ebola virus genome. The putative transcription start signal of the L gene was identical to the determined 5' terminus of the L mRNA (5' GAGGAAGAUUAA) and showed a high degree of similarity to the corresponding regions of other Ebola virus genes. The 3' end of the L mRNA terminated with 5' AUUAUAAAAAA, a sequence which is distinct from the proposed transcription termination signals of other genes. The 5' trailer sequence of the Ebola virus genomic RNA consisted of 676 nt and revealed a self-complementary sequence at the extreme end which may play an important role in virus replication. The L gene contained a single ORF encoding a polypeptide of 2212 aa. The deduced amino acid sequence showed identities of about 73 and 44% to the L proteins of Ebola virus strain Maleo (subtype Sudan) and Marburg virus, respectively. Sequence comparison studies of the Ebola virus L proteins with several corresponding proteins of other non-segmented, negative-strand RNA viruses, including Marburg viruses, confirmed a close relationship between filoviruses and members of the Paramyxovirinae. The presence of several conserved linear domains commonly found within L proteins of other members of the order Mononegavirales identified this protein as the RNA-dependent RNA polymerase of Ebola virus.

Recombinant Ebola virus nucleoprotein and glycoprotein (Gabon 94 strain) provide new tools for the detection of human infections.

After cloning and sequencing the glycoprotein (GP) gene of one of the Gabonese strains of Ebola virus isolated during the 1994-1996 outbreak, it was shown that the circulating virus was of the Zaire subtype. This was confirmed in this study by cloning and sequencing the nucleoprotein (NP) gene of this strain. These two structural proteins were also expressed as recombinant proteins and used in ELISA tests. NP was expressed as a His-tagged fusion protein in Escherichia coli and was purified on resins charged with nickel ions. GP was expressed by means of recombinant baculoviruses in Spodoptera frugiperda cells. Both recombinant proteins reacted positively in ELISAs for the detection of IgG antibodies in convalescent human sera from Gabon and Zaire. The difference in the relative titres of anti-NP and -GP antibodies was variable, depending on the sera. In addition, the recombinant NP reacted with heterologous sera from Côte d'Ivoire and was used successfully to detect IgM antibodies by mu-capture ELISA in sera from Gabonese patients.

The nonstructural small glycoprotein sGP of Ebola virus is secreted as an antiparallel-orientated homodimer.

The nonstructural small glycoprotein sGP, which unlike the transmembrane GP is synthesized from primary nonedited mRNA species, is secreted from infected cells as a disulfide-linked homodimer. Site-directed mutagenesis of all cysteine residues revealed that dimerization is due to an intermolecular disulfide linkage between cysteine residues at positions 53 and 306. Formic acid hydrolysis of sGP demonstrated that sGP dimers consist of monomers in antiparallel orientation. Another editing product of the GP gene of Ebola virus (ssGP), which shares 295 amino-terminal amino acid residues with sGP, is secreted from cells in a monomeric form due to the lack of the carboxyl-terminal part (present in sGP), including cysteine at position 306.

Release of viral glycoproteins during Ebola virus infection.

Maturation and release of the Ebola virus glycoprotein GP were studied in cells infected with either Ebola or recombinant vaccinia viruses. Significant amounts of GP were found in the culture medium in nonvirion forms. The major form represented the large subunit GP1 that was shed after release of its disulfide linkage to the smaller transmembrane subunit GP2. The minor form were intact GP1,2 complexes incorporated into virosomes. Vector-expressed GP formed spikes morphologically indistinguishable from spikes on virus particles, indicating that spike assembly is independent of other viral proteins. Analysis of a truncation mutant revealed an early and almost complete release of GP1,2 molecules, showing that membrane anchoring is mediated by the carboxy-terminal hydrophobic domain of GP2. We have also compared wild-type virus which requires transcriptional editing for synthesis of full-length GP with a variant that does not depend on editing. Both viruses released comparable amounts of GP1, but the variant expressed only minute amounts of the small, soluble GP which is the expression product of nonedited mRNA species of the GP gene. The abundant shedding of soluble GP1 may play an important role in the immunopathology of Ebola hemorrhagic fever in experimentally and naturally infected hosts.

Processing of the Ebola virus glycoprotein by the proprotein convertase furin.

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.

GP mRNA of Ebola virus is edited by the Ebola virus polymerase and by T7 and vaccinia virus polymerases.

The glycoprotein gene of Ebola virus contains a translational stop codon in the middle, thus preventing synthesis of full-length glycoprotein. Twenty percent of the mRNA isolated from Ebola virus-infected cells was shown to be edited, containing one additional nontemplate A in a stretch of seven consecutive A residues. Only the edited mRNA species encoded full-length glycoprotein, whereas the exact copies of the viral template coded for a smaller secreted glycoprotein. Expression of the glycoprotein by an in vitro transcription/translation system, by the vaccinia virus/T7 polymerase system, and by recombinant vaccinia virus revealed that full-length glycoprotein was synthesized not only when the edited glycoprotein gene (8A's) was used as a template for T7 and vaccinia virus polymerases, but also when the nonedited (genomic) glycoprotein gene was used. Analysis of mRNA produced by T7 and vaccinia virus polymerase from the 7A's construct revealed that 1-5% contained alterations at the same site that was also edited by the Ebola virus polymerase. Our data indicate that the editing site in the Ebola virus glycoprotein gene is recognized not only by Ebola virus polymerase but also by DNA-dependent RNA polymerases of different origin.

[Complete nucleotide sequence of the Eastern equine encephalomyelitis virus genome]. / Polnaia nukleotidnaia posledovatel'nost' genoma virusa vostochnogo éntsefalomielita loshadei.

The complete nucleotide sequence of the genomic 42S RNA of Eastern equine encephalitis virus has been defined for the first time. The strategy of this viral genome occurred analogous to the ones of the other alfa viruses. The comparison of amino acid sequences of E1 and E2 proteins from the two strains of the virus has revealed a number of differences. Partially, they are localized in the hydrophilic regions of the protein molecules and evidently participate in organization of the specific antigenic structures. The amino acid sequences of all viral proteins have been comparatively analysed with the sequences of the analogous proteins of other known alfa viruses.

DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition.

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.

[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. / Fragment klenova DNK-polimerazy I iz E. coli. III. Rol' mezhnukleotidnykh fosfatnykh grupp matritsy v ee sviazyvanii s fermentom.

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.