Poliovirus RNA replication requires genome circularization through a protein-protein bridge.

The mechanisms and factors involved in the replication of positive stranded RNA viruses are still unclear. Using poliovirus as a model, we show that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the viral genome is necessary for RNA replication. Initiation of negative strand RNA synthesis requires a 3' poly(A) tail. Strikingly, it also requires a cloverleaf-like RNA structure located at the other end of the genome. An RNP complex formed around the 5' cloverleaf RNA structure interacts with the poly(A) binding protein bound to the 3' poly(A) tail, thus linking the ends of the viral RNA and effectively circularizing it. Formation of this circular RNP complex is required for initiation of negative strand RNA synthesis. RNA circularization may be a general replication mechanism for positive stranded RNA viruses.     

The synthesis of minus-strand RNA of bamboo mosaic potexvirus initiates from multiple sites within the poly(A) tail

The 3' terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5' portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3' untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5' terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5' rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5'-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5' RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3' UTR of BaMV genomic RNA.     

Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules.

We have synthesized a convenient electron microscope label for mapping poly(A) sequences. Short lengths of poly(dT) are polymerized onto nicked circular SV40 DNA with the enzyme terminal deoxynucleotidyl transferase. An RNA or DNA molecule of interest is treated with glyoxal, hybridized briefly with the poly(dT) circles, and spread for microscopy; poly(A) stretches are clearly marked because they are attached to the poly(dT) on the easily recognized SV40 duplex circles. The RNAs of several type C oncornaviruses were examined by this method. The endogenous feline virus(RD-114), the endogenous baboon virus (BKD), and the woolly monkey sarcoma virus (WoMV) all contain a dimer of RNA subunits held together in a central secondary structure feature we call the dimer linkage structure. Both ends distal to the dimer linkage structure hybridize to the SV40-poly(dT). Assuming both poly(A)s are on the 3' ends of the subunits and that both subunits are identical, the two identical subunits are held together by interactions between sequences close to the 5' ends.     

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.     

Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions.

Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.     

Regions of conservation and divergence in the 3' untranslated sequences of genomic RNA from Ross River virus isolates

The 3' untranslated (UT) sequences of the genomic RNAs of five geographic variants of the alphavirus Ross River virus (RRV) were determined and compared with the 3' UT sequence of RRV T48, the prototype strain. Part of the 3' UT region of Getah virus, a close serological relative of RRV, was also sequenced. The RRV 3' UT region varies markedly in length between variants. Large deletions or insertions, sequence rearrangements and single nucleotide substitutions are observed. A sequence tract of 49 to 58 nucleotides, which is repeated as four blocks in the RRV T48 3' UT region, occurs only once in the 3' UT region of one RRV strain (NB5092), indicating that the existence of repeat sequence blocks is not essential for RRV replication. However, the precise sequence of the 3' proximal copy of the repeat block and its position relative to the poly(A) tail were identical in all RRV isolates examined, suggesting that it has an important role in RRV replication. Nucleotide substitutions between RRV variants are distributed non-randomly along the length of the 3' UT region. The sequence of 120 to 130 nucleotides adjacent to the poly(A) tail is strongly conserved. Getah virus RNA contains three repeat sequence blocks in the 3' UT region. These are similar in sequence to those in RRV RNA but differ in their arrangement. Homology between the RRV and Getah 3' UT sequences is greatest in the 3' proximal repeat sequence block that shows three differences in 49 nucleotides. The 3' proximal repeat in Getah RNA occurs at the same position, relative to the poly(A) tail, as in all RRV variants. The RRV and Getah virus 3' UT sequences show extensive homology in the region between the 3' proximal repeat and the poly(A) tail but, apart from the repeat blocks themselves, they show no significant homology elsewhere.     

The structure of poliovirus replicative form.

The structure of polio replicative form (RF) has been investigated by 3' end labeling and the use of polynucleotide phosphorylase to now allow a complete composite of the RF structure. The evidence presented indicates that the 3' terminal sequence of the minus strand is an exact complement to the 5' end of polio RNA. This suggests that the 5' terminal U of polio RNA is genetically coded. Other data is presented to show that in addition to the genetically coded poly(A) tract of the plus strand in RF, a single-stranded poly(A) tail protrudes beyond the double-stranded RNA.     

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.     

RNA-RNA recombination in Sindbis virus: roles of the 3' conserved motif, poly(A) tail, and nonviral sequences of template RNAs in polymerase recognition and template switching.

Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

A 3'' co-terminus of two early herpes simplex virus type 1 mRNAs.

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation.