Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.

Induction of intracellular membrane rearrangements by HAV proteins 2C and 2BC.

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its slow and relatively poor, noncytopathic growth in cultures of mammalian cells. The 2C and 2BC proteins of HAV have been implicated in the determination of virus growth in cultured cells. The homologous proteins from other picornaviruses, such as poliovirus, have been demonstrated to exhibit multiple activities, such as RNA binding, nucleotide binding and NTPase, and membrane binding and reorganization. At least some of these activities are required for viral RNA replication. We report here that HAV 2C and 2BC proteins, like their poliovirus counterparts, can induce rearrangement of intracellular membranes and directly or indirectly interact with membranes. Therefore, the inefficient replication properties of HAV are not consequences of the inherent ability of 2C (2BC) to interact with membranes. The effect of 2C (2BC) protein sequences derived from a cell culture-adapted (cc) strain of HAV was compared with that of corresponding protein sequences from either a wild-type (wt) strain of HAV or a faster replicating cytopathic (cp) strain. The analysis demonstrated that mutations acquired in wt virus during adaptation to cell culture do not change dramatically either the ability of these proteins to associate with membranes and induce membrane alterations or the specific architecture of the induced membrane structures. On the other hand, 2C, but not 2BC, protein from the cp strain of HAV induced different membrane structures.

Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells.

Poliovirus protein 2C is a 329-amino acid-protein that is essential for viral RNA synthesis and may perform multiple functions. In infected cells, it is associated with virus-specific membrane vesicles. Recombinant 2C protein expressed in transfected cells has been shown to associate with and induce rearrangement of the intracellular membrane network. This study was designed to map the determinants of membrane binding and rearrangement in the 2C protein. Computer-assisted analysis of the protein sequence led to a prediction that the protein folds into a structure composed of three domains. Expression plasmids that encode each or combinations of these predicted domains were used to examine the abilities of the partial protein sequences to associate with intracellular membranes and to induce rearrangement of these membranes in HeLa cells. Biochemical fractionation procedures suggested that the N-terminal region of the protein was required for membrane association. Electron microscopic and immunoelectron microscopic observation showed that both the N- and C-terminal regions, but not the central portion, of 2C protein interact with intracellular membranes and induce major changes in their morphology. The central portion, when fused to the N-terminal region, altered the specific membrane architecture induced by the N-terminal region, giving rise to vesicles resembling those observed during poliovirus infection.

Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations.

Poliovirus (PV) 2C protein is a nonstructural polypeptide involved in viral RNA replication, whose biochemical activity(ies) in this process has not been defined. By using site-directed mutagenesis, it was shown previously that disruption of nucleotide-binding motifs present in this protein abolished viral RNA synthesis (C. Mirzayan and E. Wimmer, Virology 189:547-555, 1992; N. L. Teterina, K. M. Kean, E. Gorbalenya, V. I. Agol, and M. Girard, J. Gen. Virol. 73:1977-1986, 1992). We have tested whether PV 2C or 2BC protein provided in trans could rescue the replication of these mutated genomes. Rescuing proteins were provided either by cotransfection with helper chimeric PV-coxsackievirus genomes or by expression in cells with a vaccinia virus-T7 RNA polymerase transient-expression system. We report here that replication of mutated RNAs genomes was poorly supported in trans both by helper genomes and by expressed 2C or 2BC proteins. Similarly, very inefficient complementation was observed for two mutated genomes with lethal lesions in 3D polymerase coding sequence. Our results indicate that poliovirus RNA replication shows marked preference for proteins contributed in cis.

Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein.

The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS136 and MDD177, which correspond to the consensus 'A' and 'B' motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr133 residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro131 residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys135, Ser136 or Asp177 residues. However, true revertants were selected from Arg135 and Ser135 mutants, from Glu177 and Gly177 mutants, and from Ala136 mutants. Thr136 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn140----Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphate-binding pattern in the poliovirus 2C protein.

[Non-radioactive diagnosis of viral infections using "DNA-peroxidase" probes]. / Neradioaktivnaia diagnostika virusnykh infektsii zondami "DNA-peroksidaza".

DNA-peroxidase probes were synthesized according to a modified method (Renzetal) for the detection of lambda phage DNA (model system), polio, potato X and M, tobacco mosaic viral RNAs by spot hybridization onto nitrocellulose membranes. cDNAs (300-1400 bases) complementary to the viral RNAs were cloned in M13 phage DNA or pTZ19. Efficacy of each step of the probe construction and the diagnostic procedure were thoroughly examined. Peroxidase activity manifested with non-toxic stain (NTS) was 3-5 fold more sensitive in comparison with alpha-Cl-naphthol or bisanisidine. It was found that HRP became much more stable to heat in diluted samples and was 2-3 fold more active after coupling with polyethylene imine spacer. Also, sodium borohydride reduction of the cDNA and PEI-HRP adduct crosslinked by the glutardialdehyde resulted in the stabilization of the probes. Target nucleic acids or diagnostic samples were efficiently fixed onto nitrocellulose membranes by a short-time UV irradiation. Diagnostics of cellular extracts with the preliminary prepared probes takes 4-5 hours due to express hybridization (1 hr) with 100-200 ng/ml of specific nucleotide sequence. Up to 20 pg (less than 10(-17) M) of the purified viral nucleic acids and 30-50 pg of them in the total fraction of the cellular nucleic acids isolated from the infected cells were identified with the probes. 50-10000 fold diluted lysate of the HeLa cells infected with poliovirus (PV1) and both crude extracts of potato tuber or potato and tobacco leaf tissues infected with PVX, PVM or TMV displayed specific signals with the respective DNA-HRP probes.

Analysis of putative active site residues of the poliovirus 3C protease.

It was recently suggested that the picornavirus 3C proteases are homologous to the chymotrypsin-like serine proteases. The two structural models proposed differ in one of the postulated active site residues, Glu/Asp71 or Asp85. We changed Glu71 of the poliovirus type 1 protease to Asp or Gln and Asp85 to Glu by oligonucleotide-directed site-specific mutagenesis of an infectious cDNA, and attempted to recover virus after transfection. Both Glu71 changes were lethal for the virus and proteolytic activity was abolished in vitro with the exception of the primary cleavage event at the P2/P3 junction. In contrast, the Asp85----Glu virus was viable. This mutant was temperature-sensitive for growth at 39 degrees and exhibited a minute plaque phenotype at permissive temperature. This defect correlated with low levels of viral-specific RNA and protein syntheses and slow virus growth. Proteolytic processing at the COOH-terminus of 3C was impaired, reducing the production of mature 3C and the viral replicase 3D. In addition, 3C-mediated cleavage events within the P2 region of the polyprotein seemed to occur rather inefficiently. 3C-specific processing within P1 and elsewhere within P3 was unaffected. We suggest that Asp85 does not form part of the active site of 3C, but could be important for the specific recognition of cleavage sites within P2.

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.

The nucleotide sequence of the gene coding for the 16S rRNA from the archaebacterium Halobacterium halobium.

The complete 1473-bp sequence of the 16S rRNA gene from the archaebacterium Halobacterium halobium has been determined. Alignment with the sequences of the 16S rRNA gene from the archaebacteria Halobacterium volcanii and Halococcus morrhua reveals similar degrees of homology, about 88%. Differences in the primary structures of H. halobium and eubacterial (Escherichia coli) 16S rRNA or eukaryotic (Dictyostelium discoideum) 18S rRNA are much higher, corresponding to 63% and 56% homology, respectively. A comparison of the nucleotide sequence of the H. halobium 16S rRNA with those of its archaebacterial counterparts generally confirms a secondary structure model of the RNA contained in the small subunit of the archaebacterial ribosome.

Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium.

[Topography of rRNA in ribosomes. Effect of pancreatic RNAse on small ribosomal subunits]. / Topografiia rRNK v ribosomakh. Deistvie pankreaticheskoí RNKazy na maluiu subchastitsu ribosom.

The treatment of E. coli 30S ribosome subunits with pancreatic RNase under certain conditions resulted in the release of rRNA (about 15%). The subunit retained as a whole structure: sedimentation coefficient was unchanged and no protein release was observed. The released RNA is a set of oligonucleotides from 1 to 9 bases, except hepta- and octanenucleotides. Base composition of this RNA fraction is similar to 16S RNA, a slight increase of purines content being due to the specificity of nuclease. Analysis of isoplit content has revealed that a spliting of long oligonucleotides in stechiometric amount from 30S subunits takes place: one nonanucleotide, one hexanucleotide and two pentanucleotides.