Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.     

Specific interaction between the classical swine fever virus NS5B protein and the viral genome.

The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3' untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3' UTR and that the NS5B protein was also able to bind the minus-strand 3' UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3' UTR more efficiently than the plus-strand 3' UTR. Further, we observed that the plus-strand 3' UTR with deletion of CCCGG or 21 continuous nucleotides at its 3' terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3' UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3' terminal. Therefore, it is indicated that the 3' CCCGG sequence of the plus-strand 3' UTR, and the 3' CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3' 21 nucleotide terminal site of both the 3' UTRs.     

The primary and secondary structure of the 5'-end region of encephalomyocarditis virus RNA. A novel approach to sequencing long RNA molecules

The primary structure of a 149-nucleotide fragment of encephalomyocarditis (EMC) virus RNA from the 5'-terminus of the genome up to the poly(C) tract (S fragment) has been determined. For isolation of the S fragment, site-directed fragmentation of the viral RNA with RNase H and poly(dG) was employed. For sequencing the S fragment, a novel approach has been developed, which can be used for primary structure determination of long RNA molecules. A model of the secondary structure of the S fragment is proposed, according to which this region of RNA is highly structured. The role of complementary oligonucleotide stretches near both termini of the RNA molecule is discussed.     

Cloning of the hemagglutinin gene of influenza virus of subtype H3 in E. coli

dsDNA of the influenza virus subtype A/Leningrad/385/80/R (H3N2)-recombinant A/Leningrad/385/80 (H3N2) and RR/8/34 (H1N1) has been synthesized using polyadenylated viral RNA as a template. This dsDNA has been cloned on plasmid pUC19. A clone has been selected harbouring the plasmid with included proximal fragment of hemagglutinin gene that contains the main antigenic determinants. The hybrid plasmid is hybridizable with RNA of the hemagglutinin gene and with oligonucleotide CATGCAAAACCTTCCC that is complementing the sequence coding for the proximal fragment of the mature hemagglutinin.     

The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.     

The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3' long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.     

Mappings of adenovirus type 7 cytoplasmic RNA species synthesized early in lytically infected cells and synthesized in transformed cells.

Early virus-specific RNA synthesized in KB cells infected with adenovirus type 7 and virus-specific RNA synthesized in rat embryo cells (71JY1-2) transformed by the adenovirus type 7 HindIII-I.J fragment (left-hand 8.1% of the viral genome) have been mapped on the viral genome. About 25% of the viral genome, four discrete regions, two on each strand of the viral genome, are expressed as "early" mRNA. Almost similar regions in the left-hand 8.1% of the viral genome are transcribed both in KB cells at early times after infection and in 71JY1-2 cells.     

Use of bromovirus RNA2 hybrids to map cis- and trans-acting functions in a conserved RNA replication gene.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with tripartite genomes. RNA replication by either virus requires genomic RNAs 1 and 2, which encode protein 1a and the polymeraselike, 94-kilodalton 2a protein, respectively. Proteins 1a and 2a share extensive sequence similarity with proteins encoded by a wide range of other positive-strand RNA viruses of animals and plants. Heterologous combinations of BMV and CCMV RNAs 1 and 2 do not support viral RNA replication, and although BMV RNA2 is amplified in CCMV-infected cells, CCMV RNA2 is not amplified by BMV. Construction of hybrids by precise exchange of segments between BMV and CCMV RNA2 has now allowed preliminary mapping of such virus-specific replication functions in RNA2 and the 2a protein. The ability to support replication in trans with BMV RNA1 segregated with a 5' BMV RNA2 fragment encoding the first 358 2a gene amino acids, while a 5' fragment extending over 281 BMV 2a codons transferred only cis-acting competence for RNA2 amplification in cells coinfected with wild-type BMV. Successful trans-acting function with CCMV RNA1 segregated with a CCMV RNA2 3' fragment that included the last 206 2a gene codons. Thus, the less conserved N- and C-terminal 2a segments appear to be involved in required interaction(s) of this polymeraselike protein with the 1a protein or RNA1 or both. Moreover, when individual hybrid RNA2 molecules that function with either BMV or CCMV RNA1 were tested, BMV- and CCMV-specific differences in recognition and amplification of RNA3 templates appeared to segregate with RNA1.     

Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.