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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.

The structure of the Fe18%Cr alloys with 0.6, 0.9, 1.1 and 1.3%N quenched from different temperatures was studied using electron microscopy and X-ray analysis. It was shown that the temperature of quenching and N concentration in alloys determine which structure is formed after quenching: austenite only, austenite + martensite; austenite + CrN or ferrite + CrN. The type of the quenched structure determines the character of the alloys decomposition during ageing. The decomposition of the austenite begins with CrN mixed zones formation and simultaneously γ-α transformation. Further, the mixed zones transform into CrN with a tetragonally distorted B1 structure of the NaCl type. Nitrides present in the quenched structure (martensite or ferrite) result in further coarsening during ageing.

The structure of an iron alloy containing 18% Cr and 0.72% N after different treatments was studied by X-ray and electron microscopy. It was shown that the ψ-structure of the alloy after quenching is non-stable at all temperatures of isothermal holding, especially at high tempering temperatures. The C-shaped hypothetical diagram of the alloy was constructed from the experimental results.

The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.

An analysis of reported nucleotide sequences revealed several cases of gross rearrangements in the 5'-untranslated region (5-UTR) of picornaviral genomes. A large (greater than 100 nt) duplication was discovered in a downstream region of poliovirus 5-UTR involved in the translational control. Properties of the poliovirus mutants with large deletions [Kuge and Nomoto (1987) J. Virol. 61, 1478-1487] show that a single copy of the appropriate repeating unit is compatible with a wild type phenotype of the virus. In contrast to poliovirus and another enterovirus genomes, human rhinovirus RNAs contain only a single copy of this repeating unit. Another similarly large repeat was found in an upstream segment of the bovine enterovirus 5-UTR. A comparison of the primary and secondary structures of cardio- and aphthovirus 5-UTRs demonstrated the existence of a large (ca. 250 nucleotides) insertion/deletion in a region preceding the poly(C) tract. The two latter rearrangements appear to involve elements of the viral genome replication machinery. Possible origin as well as evolutionary and functional implications of these structural peculiarities are discussed.

Computer analysis of full coding sequences of variola major virus strain India-1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin-like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.

A package for the creation and processing of multiple sequence alignment is described. There is no limit on the lenghts of the processed nucleotide or amino acid sequences, and the number of sequences in the alignment is also unlimited. The main groups of functions are: a semi-automatic alignment editor; a wide set of functions for technical processing of alignments; nucleotide alignment mapping and translation; and similarity search functions. A user-friendly interface and a set of generally used file actions provide a special operational subsystem for everyday tasks

Genomic RNA of a Zaire strain of Ebola virus was cloned, and cDNA inserts specific for the glycoprotein gene were isolated and sequenced. The determined sequence has only one open reading frame encoding 318 amino acids and is part of ORF-4 on the plus RNA strand. The putative transcriptional stop site (3′ AAUUCUUUUU 5′) and the transcriptional start site (3′ AACUACUUCUAAUU.. 5′) were identified. Computer-assisted comparison of the amino acid sequence of the C-terminal part of protein encoded by ORF-4 of Ebola virus with sequence of the proteins present in the SWISSPROT and EMBL banks revealed significant homology with the immunosuppressive domain of the p15E envelope proteins of various oncogenic retroviruses. The possible role of such a homology is discussed.

The initiation sites of the Galleria mellonella L. nuclear polyhedrosis virus (G.m. NPV) DNA replication were revealed. For this purpose SCLd 135 cells permitting the G.m. NPV productive reproduction were transformed by the recombinant plasmids containing the viral genome individual fragments in pRSF 2124 and pBR 322 vectors. It was revealed that 2 of the 32 recombinant plasmids can autonomously replicate in the eucaryotic cells. According to the Maxam-Gilbert method the DNA G.m. NPV fragment (1300 bp) primary structure of pHBR plasmid was determined. The structure analysis revealed the typical regulator signals as in the replicons. The possible regulation mechanisms of the DNA G.m. NPV synthesis initiation was supposed.

A statistically significant similarity was demonstrated between the amino acid sequences of 4 Escherichia coli helicases and helicase subunits, a family of non-structural proteins of eukaryotic positive-strand RNA viruses and 2 herpesvirus proteins all of which contain an NTP-binding sequence motif. Based on sequence analysis and secondary structure predictions, a generalized structural model for the ATP-binding core is proposed. It is suggested that all these proteins constitute a superfamily of helicases (or helicase subunits) involved in NTP-dependent duplex unwinding during DNA and RNA replication and recombination.

Recently we tentatively identified, by sequence comparison, central domains of the NS3 proteins of flaviviruses and the respective portion of the pestivirus polyprotein as RNA helicases (A.E.G. et al., submitted). Alignment of the N-proximal domains of the same proteins revealed conservation of short sequence stretches resembling those around the catalytic Ser, His and Asp residues of chymotrypsin-like proteases. A statistically significant similarity has been detected between the sequences of these domains and those of the C-terminal serine protease domains of alphavirus capsid proteins. It is suggested that flavivirus NS3 and the respective pestivirus protein contain at least two functional domains, the N-proximal protease and the C-proximal helicase one. The protease domain is probably involved in the processing of viral non-structural proteins.

Summary NTP-motif, a consensus sequence previously shown to be characteristic of numerous NTP-utilizing enzymes, was identified in nonstructural proteins of several groups of positive-strand RNA viruses. These groups include picorna-, alpha-, and coronaviruses infecting animals and como-, poty-, tobamo-, tricorna-, hordei-, and furoviruses of plants, totalling 21 viruses. It has been demonstrated that the viral NTP-motif-containing proteins constitute three distinct families, the sequences within each family being similar to each other at a statistically highly significant level. A lower, but still valid similarity has also been revealed between the families. An overall alignment has been generated, which includes several highly conserved sequence stretches. The two most prominent of the latter contain the socalled “A” and “B” sites of the NTP-motif, with four of the five invariant amino acid residues observed within these sequences. These observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the NTP-motif-containing proteins of positive-strand RNA viruses are homologous, constituting a highly diverged monophyletic group. In this group the “A” and “B” sites of the NTP-motif are the most conserved sequences and, by inference, should play the principal role in the functioning of the proteins. A hypothesis is proposed that all these proteins posses NTP-binding capacity and possibly NTPase activity, performing some NTP-dependent function in viral RNA replication. The importance of phylogenetic analysis for the assessment of the significance of the occurrence of the NTP-motif (and of sequence motifs of this sort in general) in proteins is emphasized.

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal β-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.

It is demonstrated, by computer-assisted analysis, that T5 bacteriophage early genes D10 and D13 encode proteins containing the purine NTP-binding sequence motif. The D10 gene product is shown to be a member of a recently characterized superfamily of (putative) DNA and RNA helicases. The D13 gene product is related, at a statistically significant level, to the gene 46 product of bacteriophage T4 which is a component of an exonuclease involved in phage DNA replication, recombination and repair. A lower but also significant degree of sequence similarity was detected between the gene D12 product of T5 and the gene 47 product of T4, the second component of the same nuclease. It is hypothesized that both D10 and D13 gene products of T5 might be NTPases, possibly DNA-dependent, mediating NTP-consuming steps during phage DNA replication, recombination and/or repair.

RNA of a flavivirus, tick-borne encephalitis virus (TBEV; strain Sofjin), was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by the action of E. coli DNA-polymerase I (Klenow fragment). This DNA was annealed with plasmid pBR322. The recombinant plasmids were cloned in E. coli K802. The nucleotide sequence of the inserts of the clones, coding for region structural proteins C, M, E and nonstructural protein NS1, was determined by the Maxam-Gilbert method. The genes of structural proteins form a compact cluster. Homology has been studied of the TBEV sequences found with the structures of proteins and RNAs of other flaviviruses, yellow fever virus and West Nile virus, and a high degree of homology was found.

cDNA was synthesized and cloned on the template of the genomic RNA of Marburg virus (strain Popp). Recombinant plasmids with specific cDNA inserts were selected and sequenced. The length of the open reading frame encoding the GP-protein is 681 amino acids. GP-protein is proposed to be an integral membrane protein. Computer-assisted comparison of the deduced amino acid sequence with those of different viruses revealed significant homology with the GP-protein of Ebola virus and with the ‘immunosuppressive domain” of the P15E envelope proteins of some oncogenic retroviruses.

Analysis of variola virus nucleotide sequence revealed proteins belonging to several families which provide the virus with the possibility of overcoming the barriers of specific and non-specific host defence against viral infection. The complement-binding proteins, lymphokine-binding proteins, and serine protease inhibitors can be assigned to this type, as can the proteins providing the orthopoxviruses with resistance to interferon. The revealed differences between the genes (proteins) of variola and vaccinia viruses under study are discussed.

The complete genetic map of the variola major virus strain India-1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.

To elucidate the mode of action of norakin against influenza A virus we sequenced the hemagglutinin gene of 11 norakin-resistant mutants. Resistance was coupled with 1–3 amino acid exchanges. The majority of mutations was localized in the HA2 polypeptide and was mostly associated with changes in charge or polarity of the amino acids. The amino acid substitutions are discussed in the context of the 3D structure of X31 hemagglutinin considered to be representative of the influenza hemagglutinins. Most of the mutations appear to destabilize the pH 7.0 structure by distorting or destroying hydrogen bonds as well as salt-bridges which are responsible for intra- and intersubunit contacts, while others destabilize the location of the fusion peptide, facilitating conformational changes in the presence of the inhibitor.

Here we demonstrate significant similarities between the amino acid sequences of trypsin (a serine protease) and the N-terminal piece of a specific fragment of the poliovirus polyprotein encompassing the sequence of the viral proteinase 3C, and also between cathepsin H (a cysteine protease) and the C-terminal piece of the same fragment. A coherent alignment of the sequences of the 3 proteases was obtained, in which the principal catalytically active residues occupy identical positions. A hypothesis is proposed that the viral enzyme may provide an evolutionary link between serine and cysteine protease families.

In the course of systematic analysis of protein sequences containing the purine NTP-binding motif, a new superfamily was delineated which included 25 established or putative helicases of Escherichia coli, yeast, insects, mammals, pox- and herpesviruses, a yeast mitochondrial plasmid and three groups of positive strand RNA viruses. These proteins contained 7 distinct highly conserved segments two of which corresponded to the "A" and "B" sites of the NTP-binding motif. Typical of the new superfamily was an abridged consensus for the "A" site, GxGKS/T, instead of the classical G/AxxxxGKS/T. Secondary structure predictions indicated that each of the conserved segments might constitute a separate structural unit centering at a beta-turn. All previously characterized mutations impairing the function of the yeast helicase RAD3 in DNA repair mapped to one of the conserved segments. A degree of similarity was revealed between the consensus pattern of conserved amino acid residues derived for the new superfamily and that of another recently described protein superfamily including a different set of prokaryotic, eukaryotic and viral (putative) helicases.

Amino acid sequences of 2 giant non-structural polyproteins (F1 and F2) of infectious bronchitis virus (IBV), a member of Coronaviridae, were compared, by computer-assisted methods, to sequences of a number of other positive strand RNA viral and cellular proteins. By this approach, juxtaposed putative RNA-dependent RNA polymerase, nucleic acid binding ("finger"-like) and RNA helicase domains were identified in F2. Together, these domains might constitute the core of the protein complex involved in the primer-dependent transcription, replication and recombination of coronaviruses. In F1, two cysteine protease-like domains and a growth factor-like one were revealed. One of the putative proteases of IBV is similar to 3C proteases of picornaviruses and related enzymes of como- nepo- and potyviruses. Search of IBV F1 and F2 sequences for sites similar to those cleaved by the latter proteases and intercomparison of the surrounding sequence stretches revealed 13 dipeptides Q/S(G) which are probably cleaved by the coronavirus 3C-like protease. Based on these observations, a partial tentative scheme for the functional organization and expression strategy of the non-structural polyproteins of IBV was proposed. It implies that, despite the general similarity to other positive strand RNA viruses, and particularly to potyviruses, coronaviruses possess a number of unique structural and functional features.

A putative serine protease was identified among non-structural proteins of southern bean mosaic virus (SBMV) by sequence comparison with cellular and viral proteases. The predicted SBMV proteased is played a significant similarity to cysteine proteases of picornaviruses, providing a possible evolutionary link between the two enzyme classes. It is suggested that SBMV follows the general expression strategy characteristic of other positive-strand RNA viruses containing 5′-terminal covalently linked proteins (VPg), i.e. generation of functional proteins by polyprotein processing.