[Comparison of the homologous SfeI and LlaBI restriction-modification systems]. / Sravnitel'nyi analiz gomologichnykh sistem restriktsii-modifikatsii SfeI i LlaBI.

A fragment containing the SfeI restriction-modification system (RMS) operon was cloned from a Streptococcus faecalis SE72 plasmid. Nucleotide sequence analysis revealed its high (99.2%) homology to the operon for Lactococcus lactis subsp. cremoris W56 LlaBI RMS recognizing the same site, 5'-CTRYAG-3'. A substantial difference was that SfeI RMS operon had an additional 198-bp fragment and a larger gene for the putative control protein. No homology was observed between operon-flanking sequences of the two closely related species, suggesting horizontal transfer of the operon.

The genome of hawaii virus and its relationship with other members of the caliciviridae.

Hawaii virus (Hu/NLV/GII/Hawaii virus/1971/US), a member of the genus 'Norwalk-like viruses' (NLVs) in the family Caliciviridae, has served as one of the reference strains for the fastidious caliciviruses associated with epidemic gastroenteritis in humans. The consensus sequence of the RNA genome of Hawaii virus was determined in order to establish its relatedness with other members of the family. The RNA genome is 7,513 nucleotides (nts) in length, excluding the 3'-end poly (A) tract, and is organized into three major open reading frames (ORFI, nts 5-5,104; ORF2, nts 5,085-6,692; and ORF3, nts 6,692-7,471). Phylogenetic analysis showed the closest relatedness of Hawaii virus throughout its genome to Lordsdale virus, a Genogroup II NLV. Analysis of the predicted secondary structure of the RNA from the 5'-end of the genome and the putative beginning of the subgenomic RNA showed the presence of two hairpin structures at both ends that are similar to each other and to those of other NLVs.

Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase.

The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

Identification and genomic mapping of the ORF3 and VPg proteins in feline calicivirus virions.

Two minor proteins with molecular masses of 8.5 and 15.5 kDa were identified in feline calicivirus (FCV) virions. Direct sequence analysis showed that the N-terminal sequence of the 8.5-kDa protein was identical to that of the predicted protein encoded by open reading frame 3 (ORF3) of the FCV genome. The N-terminal sequence of the 15.5-kDa protein corresponded to amino acids 961-980 of the FCV ORF1 polyprotein and mapped to the genomic location of the calicivirus VPg. Antisera raised against recombinant ORF3 protein or the N-terminal 20 amino acids of the putative VPg reacted with the corresponding proteins present in both a Western blot analysis of purified FCV virions and an immunofluorescence assay of FCV-infected cells. A comparative analysis of radioactivity incorporated into virion proteins during in vivo labeling experiments indicated that the ORF3 protein is likely present in one or two copies per virion. The mobility of the ORF3 protein present in virions was similar to that of the ORF3 protein found in FCV-infected cells or expressed in bacteria. Direct N- and C-terminal sequence analysis of the purified ORF3 protein obtained by expression in bacteria demonstrated the presence of intact, uncleaved termini, suggesting that the observed difference between the calculated and the apparent masses in SDS-PAGE was not due to proteolytic processing of the protein.

Recovery and altered neutralization specificities of chimeric viruses containing capsid protein domain exchanges from antigenically distinct strains of feline calicivirus.

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization.

Mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein.

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.

Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells.

The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells. Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression. The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells. There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein. Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study.

Cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase.

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3'-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1' position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.

RNA transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require VpG for infectivity.

Feline calicivirus (FCV) is a positive-strand, nonenveloped RNA virus in the family Caliciviridae. A cDNA library of the Urbana (URB) strain of FCV was generated and the sequence of the genome was determined from overlapping clones except for 13 bases from the 5'-end. The 5'-end sequence was identified by analysis of clones derived by RT-PCR across the ligated 5'- and 3'-ends of the RNA genome. A full-length cDNA clone of the RNA genome of the URB strain was constructed and placed downstream of the T7 RNA polymerase promoter and RNA transcripts generated in vitro from this clone were infectious when introduced into feline kidney cells. A virus-encoded genome-linked protein, VpG, which is considered to be essential for infectivity of wild-type genomic FCV RNA, was not required for the initiation of FCV infection by the synthetic transcripts. However, the addition of a cap structure analog (m7G(5')ppp(5')G) during in vitro transcription of the synthetic RNA was necessary for successful virus recovery. Two silent mutations engineered into the full-length clone were identified in the genomic RNA from recovered progeny virus. This system of introducing site-specific genetic changes into the genome of feline calicivirus and the recovery of infectious mutant viruses will enable studies related to the molecular basis for replication, growth restriction, and pathogenicity of this and other members of the Caliciviridae.

[Modular evolution of Bsp-repeats: formation of subrepeats and monomers preceded the divergence of four species of Canidae]. / Blochnaia évoliutsiia Bsp-povtorov: formirovanie subpovtorov i monomerov predshestvovalo raskhozhdeniiu chetyrekh vidov Canidae.

Determination of two nucleotide sequences of satellite-like DNA (Bsp-repeats) segments from the raccoon-like dog genome and analysis of these and three other homologous sequences from distantly related genomes of Canidae (silver fox, domestic dog, and gray fox) show that all these DNA fragments consist of five types of subrepeats (A, B, C, D, and E) and two types of monomers-ACB and ACED. Raccoon-like dog repeated segment of 1620 bp is composed of both type of monomers: ACBACED. Different monomers of the same type could be distinguished by subtypes of subrepeats. Building of a cladogram of 18 subrepeats and analysis of the structures of monomers and higher-order repeated units allowed us to reveal probable evolution events which have determined the hierarchical order in Bsp-repeat organization. Approximate periods of time for these evolution events were calculated from the number of substitutions per branch. Five main types of subrepeats and two types of monomers have been generated and propagated through several steps before divergence of the main phylogenetic trees of Canidae. Combinatorial variability appears to play a fundamental role in forming the hierarchical layers in Bsp-repeats structure.

[The isolation and study of the characteristics of a cytopathic strain of the hepatitis A virus]. / Vydelenie i izuchenie kharakteristik tsitopaticheskogo shtamma virusa gepatita A.

A fast-growing cytopathic isolate of human hepatitis A virus (strain MB-7) was derived from fecal samples of infected patients and adapted to growth in FRhK-4 cell culture. A positive serum standard against HAV and electron microscopy were used to demonstrate that MB-7 belonged to human hepatitis A virus. The strain MB-7 induced plaque formation in FRhK-4 under agar overlay after 10-12 days of incubation. The PCR products of gene VP1 were cloned in E. coli and its primary structure was determined. MB-7 was shown to have more homology with HAV strains isolated in the USA and China.

[Features of the structure and evolution of complex tandemly organized Bsp-repeats in the fox genome. II. Tissue-specific and recombinant BamHI-dimer sites]. / Osobennosti struktury i évoliutsii slozhnykh tandemno organizovannykh Bsp-povtorob genoma lisitsy. II. Tkanespetsificheskie i rekombinatsionnye saity BamHI-dimera.

The complex structure of the clustered Bsp-repeats in fox genome seems to have evolved throughout a long period of time as a result of multiplication, recombination and divergence events. The sequence of the subrepeat (SR) approximately 245 b.p long is the basic substructure for the hierarchically arranged Bam HI-repeat 1468 b.p. long. The monomer consists of 3 SRs with a 43-59% homology. A dimer is composed of 2 monomers with a 93% homology. Amplification of the Bsp-repeats during evolution seems to have occurred at least twice: first--on the SR ancestral form level, second--on the monomer level. Despite profound divergence, there are still conservative regions in SRs with sequences homologous to known functional sites in eukaryotes. However qualitative and quantitative composition of most functional motifs is stringently individual in every SR. The performed analysis revealed that throughout evolution SRs acquired significant amount of motifs homologous to promoter and enhancer regions in tissue-specific genes and virus regulatory regions. Functional motifs in separate SRs are being differently grouped. Most inducible motifs are located in the III and II subrepeats, putative promoters--in the II one; elements participating both in transcriptional and replicational processes--mainly in the I subrepeat. A few ensembles of functional motifs remotely resemble extended regulatory regions of some tissue-specific genes. The monomers are potentially capable of ensuring diverse aspects of transcriptional regulation. As a whole, motifs of the 3 SRs are potentially capable of regulating the RNA synthesis periodicity with respect to the cellular cycle, activation and repression of genetical material in response to signals from the environment (AP-1, AP-2, AP-4, T-antigen, etc) and temporal ("octamers") etc. Apart from the BamHI-dimer, a few homologues fragments were isolated from fox genome and sequenced. Some of them were rearranged with respect to the BamHI-dimer. Inversion locally alters the composition of motifs and the sequence acquires new functional potential. Thus, the analysis of the emergence and development of Bsp-repeat structural variations allows us to consider repetitive DNA sequences as an ideal material in constructing multiprofile regulatory sequences.

[Features of the structure and evolution of complex, tandemly organized Bsp-repeats in the fox genome. I. Structure and internal organization of the BamHI-dimer]. / Osobennosti struktury i évoliutsii slozhnykh, tandemno organizovannykh, Bsp-povtorov genoma lisitsy. I. STruktura i vnutrenniaia organizatsiia BamHI-dimera.

A 1468 b.p. DNA BamHI-fragment homologous to the Bsp-repeat was isolated from the fox genome and sequenced. This fragment is an hierarchically arranged dimer. Its 734 b.p.-monomers consist of three subrepeats (SR), each 245 b.p. long, abundant with overlapping imperfect tandem repeats which in turn are rich in short direct related repeats (each 4-7 b.p. in size). The latters are mainly composed of AG, TG dinucleotides and their complements CT, CA. All subrepeats in the BamHI-dimer are flanked by motifs homologous to Jeffreys' sites. At certain points the sites are doubled. The above data allow to assume that the Bsp-repeat complex structure is likely to have developed throughout long multi-step evolution of relatively simple DNA sequences which had emerged de novo. Single substitutions, small inserts and deletions, multiple duplication and recombination events seem to have most contributed to the evolution of the Bsp-repeats. Single substitutions in SRs with respect to the consensus are not equally distributed along their length. A wave-like pattern of this distribution is the evidence for non-random character of mutations accumulation. A correspondence was noted between conservative regions in SR and the presence therein of functional motifs homologous to the binding sites of already known regulatory proteins.

[Primary structure of the gene for protein VP1 from foot-and-mouth disease virus serotype O1 isolated in the USSR]. / Pervichnaia struktura gena belka VP1 virusov iashchura serotipa O1, izolirovannykh v SSSR.

The nucleotides sequence has been determined for the viral RNA and some of its cDNAs coding for the major antigenic protein of the epidemic stomatitis O1-194 and O1-1618 VP1 viruses. The expressed microheterogeneity has been registered for the population of the strain O1-194.

[Primary structure of the gene for VP1 protein of the foot-and-mouth disease virus of Asia 1 serotype]. / Pervichnaia struktura gena belka VP1 virusa iashchura serotipa Aziia 1.

The nucleotide sequence of the cDNA for the viral RNA region coding for the main antigenic protein of the epidemic stomatitis virus of Asia 1 serotype has been identified. The amino acid sequences in the regions of VP1 protein antigenic determinants of the serotype Asia 1 virus and other serotypes viruses have been compared.

[Organization of Bsp-repeats in the fox genome]. / Organizatsiia Bsp-povtorov v genome lisitsy.

Bsp repeats comprise approximately 1% of silver for total DNA and are preferentially localized in centromeric regions. Two of Bsp fragments cloned earlier, such as non-homologous rsV1 and rsV3, make up together a Bsp unit (680 bp) and possess a set of sites known to have regulatory functions in eucaryotic genomes. In this work, tandem organization of Bsp repeats is demonstrated. A stretched Bsp sequence (approximately 1460 bp, dimeric form) flanked by BamHI sites was cloned and its restriction map was plotted. With use of rsV1 and rsV3 probes the new sequence exhibited linked structure: rsV1-rsV3-rsV1-rsV3-rsV1. Blot-hybridization with the restriction fragments of fox total DNA suggested hierarchical order of Bsp clustes in the genome. It is supposed that the processes of selective amplification of individual fragments had been of real importance during evolution of Bsp repeats.