Structure and origin of a snapback defective interfering particle RNA of vesicular stomatitis virus

The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.     

Defective interfering particle generated by internal deletion of the vesicular stomatitis virus genome.

The genome structure of the long, truncated defective interfering particle derived from the heat-resistant strain of vesicular stomatitis virus has been examined. Stocks of this defective interfering particle are shown to contain several different species having information primarily from the 3' half of the vesicular stomatitis virus genome; the proportions of these components vary depending on the passage history of the stock. The two most abundant types have been identified and characterized. One has complementary 5' and 3' termini and consequently appears as a circular molecule when examined by electron microscopy. The other cannot circularize and remains linear. The circular forms are consistently 8 to 10% longer than the linear molecules. Rapid sequencing analyses reveal that both forms retain the 5' parental viral terminal sequence, but only the linear form retains the parental 3'-terminal sequence which is the complement of the 5' end. Hybridization experiments and electron microscopic analyses indicate that the linear form has retained 320 to 350 nucleotides of the 5' parental sequence and was probably generated by an internal deletion of the vesicular stomatitis virus genome.     

Structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus

The genome structure and terminal sequences of a 'copyback' defective interfering (DI) particle ST1, and a novel complexly rearranged 'snapback' DI particle ST2 of vesicular stomatitis virus have been determined. The ST1 DI genome RNA possesses 54 base long inverted complementary termini, the 5' end of which is homologous to the standard virus genome 5' end. Following this region of inverted complementarity the DI RNA 5' end continues to be homologous to standard virus RNA 5' sequences, whereas the 3' end diverges into sequences within the virus L gene internal sequences. ST2 DI genome RNA does not contain colinear covalently linked plus and minus sense RNA copies of the standard infectious virus RNA 5' terminus as predicted from the prototype snapback DI structure, but instead appears to be a hairpin copy of the ST1 DI RNA genome. This is the first evidence suggesting that DI particles may be generated from RNA templates other than the standard virus RNA. Generation models and the implications of these findings for RNA virus evolution are discussed.     

Nucleotide sequences of influenza virus segments 1 and 3 reveal mosaic structure of a small viral RNA segment

Defective interfering RNAs of influenza virus are small segments derived from viral segments 1, 2 and 3. We present here the complete nucleotide sequences of segments 1 and 3 from the human influenza strain A/PR/8/34 and deduce that the sequence of a small RNA segment from A/NT/60/68, apparently a defective interfering RNA, is derived from five separate regions in segment 3 and from one region in segment 1. These regions, which are located near the termini of the two parental segments, are arranged in the small RNA segment in an alternating fashion: thus a region derived from near a 5′ terminus is adjacent to a region derived from near a 3′ terminus. We propose that the small segment is generated during positive strand synthesis as a result of the viral polymerase pausing at uridine-rich sequences in the template and reinitiating synthesis at another site.     

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase alpha in vitro was analyzed using circular, single-stranded M13 or phi X174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3'-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by alpha-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.     

DNA sequence analysis of the defective interfering particles of bacteriophage f1.

Restriction mapping and nucleotide sequence analysis of several defective, interfering particles of bacteriophage f1 are described. These particles contain the nucleotide sequences corresponding to the carboxyl terminus of gene IV and the amino-terminus of gene II and the intergenic space between them. Tandem duplication of a portion of this intergenic space generates defective particles with novel nucleotide sequences not found in wild-type f1. This duplication is shown to contain the origin of complementary strand synthesis. Our results suggest that the duplication occurs at the site of gene II protein action, i.e. the origin of viral strand synthesis. A model is presented for the generation of these duplications in defective particles.     

In vitro construction of poliovirus defective interfering particles.

To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase. The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion. HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper. The DI RNA was observed in the infected cells after three passages at high multiplicity of infection. The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection. Thus, we succeeded in construction of a poliovirus DI particle in vitro. To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978. The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon. On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells. Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a. These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s). The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.     

The nucleotide sequence of the 5'' terminus of vesicular stomatitis virus RNA.

We have determined the nucleotide sequence for the first 50 nucleotides at the 5' terminus of vesicular stomatitis virus (VSV) genome RNA. This sequence is identical to that of the in vitro RNA polymerase product synthesized by defective interfering (DI) particles of VSV. These results confirm previous conclusions rengarding DI and standard viral terminal sequences based on hybridization studies and earlier sequencing of the DI polymerase product RNA.