Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.     

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.     

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.     

The role of the OOP antisense RNA in coliphage λ development

We have made a derivative of bacteriophage lambda that makes no OOP antisense RNA. The mutant phage carries a point mutation that inactivates the OOP promoter, po. The phages lambda + and lambda po- have identical plaque morphologies, one-step growth curves, and frequencies of lysogenization of a sensitive host. OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein. Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two-fold greater phage burst following ultraviolet induction of a lambda + than of a lambda po- prophage. In lambda + infections, OOP RNA causes two cleavage events in cll mRNA: one is in the 3'-end of the coding region, and the second is in the intercistronic region between the cll and O genes. The cll gene fragments are subject to additional hydrolytic events, and cll mRNA levels are several-fold lower in lambda + than in lambda po- infections late in the infection cycle. However, O mRNA levels are almost unaffected by the po- mutation.     

Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.     

A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA

Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5'- and 3'-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5'- and 3'-TR present in the same template molecule or when the 5'-TR RNA was added in trans to the 3'-untranslated region (UTR) RNA. However, the 3'-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3'-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5'- and 3'-TR of the viral RNA that modulates the 3'-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction.     

Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA.

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.     

Interference with viral infection by RNA replicase deleted at the carboxy-terminal region

Escherichia coli cells harboring an altered Q beta RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr356-Gly357-Asp358-Asp359 of the beta-subunit protein lost the replicase activity but interfered with proliferation of Q beta phage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the beta-subunit protein. The cells expressing the beta-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Q beta phage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the beta-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Chemical synthesis of the 5'-terminal structure of U1 RNA

A new method for the synthesis of N2,N2-dimethylguanosine (DMG) is developed by reductive C-S bond cleavage by use of tributyltin hydride. An improved method for the synthesis of the key intermediate (1) for construction of the 5'-terminal structure of U1 RNA, which has a trimethylated cap (TMG) structure at its 5' end, is also described. By the use of 1, several TMG-capped ribonucleosides and oligonucleotides were synthesized.     

A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.     

Use of antisense RNA to help identify a genomic clone for the 5' region of mouse beta-glucuronidase

It was possible to gauge the inhibition of mouse beta-glucuronidase expression by injecting RNA, made from both strands of subclones of a cosmid containing the complete gene, into mouse blastomeres at the four-cell stage. Although our initial screen did not identify the 5' region, we were able to isolate a subclone containing homology to 20 bp coding for N-terminal amino acids of rat and human beta-glucuronidase structural genes. Antisense RNA prepared from one strand of the 350 bp Pst I subclone inhibited beta-glucuronidase expression by 89% while RNA prepared from the other strand had little effect. The subclone appears to correspond to the 350 bp fragment identified by others as one including the ATG start site of mouse beta-glucuronidase.     

Secondary structure of the 5'-terminal region of the 18S ribosomal RNA5.3S fragment from the rat liver

Two small RNA fragments, 5,3S and 4,7S, were observed in gel electrophoretic analysis of RNA of the 40S ribosomal subunit of rat liver. 5,3S RNA (134-136 nucleotides long) proved to be 5'-terminal fragment of 18S ribosomal RNA, whereas 4,7 RNA is the degradation product of 5,3S RNA with 27-28 5'-terminal nucleotides lost. The secondary structure of 5,3S RNA was probed with two structure-specific nucleases, S1 nuclease and the double-strand specific cobra venom endoribonuclease. The nuclease digestion data agree well with the computer generated secondary structure model for 5,3S RNA. This model predicts that the 5'-terminal part of rat liver ribosomal 18S RNA forms an independent structural domain. The affinity chromatography experiments with the immobilized 5,3S fragment show that 5,3S RNA does not bind rat liver ribosomal proteins.