Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.

Abstract

The 3'-and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-GU-A-G-A-A-AC-A-A-G-G-... -C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenza virus are formed by an internal deletion of the genomic RNA.Defective interfering (DI) animal viruses are noninfectious viruses that interfere with the replication of standard viruses (1, 2). DI influenza virus is produced after repeated undiluted passage of the virus (3), and over 99% of the infectious virus population is replaced by DI virus (4). The DI influenza virus has small virus-specific RNA molecules not present in the standard virus (4-7). Recently it has been directly demonstrated that the ribonucleoprotein complexes of these new deleted forms of the viral RNA specifically cause interference (8).In a previous report (9) the sequence relationships among these DI-RNAs and their relationship to the standard eight viral genes were studied by oligonucleotide mapping. Because each clone of influenza virus produces a unique set of DI RNAs when passaged (4), several DI RNAs are present in each DI virus preparation. All the DI RNAs studied thus far (9) have been found to be forms of one of the viral polymerase genes (P1, P2, or P3) from which sequences have been deleted. Comparison of the oligonucleotide maps of DI RNAs and P genes revealed that the DI RNAs originating from the same polymerase gene were related in one of two ways. Either they contained completely overlapping regions or each contained both overlapping and nonoverlapping regions (9). This latter group of DI RNAs could not be formed from the progenitor RNA by a mechanism of common initiation and simple deletion from one end as reported for vesicular stomatis virus (10) and Sendai virus (11). We therefore proposed (9) internal deletion of the progenitor virion RNA as one possible model for the formation of influenza DI RNAs.We have now determined the sequences of the 3' and 5' ends of three DI RNAs present in a DI influenza virus preparation as well as the sequences of the 3' and 5' ends of the polymerase genes. The sequence analysis of the DI RNAs and their progenitor genes indicates a model for the formation of influenza DI RNAs quite different from that proposed for other negative-stranded viruses (1, 11). (Mr 1 X 106). The third, Li, is also a deleted form of the P3 gene (9). MATERIALS AND METHODSPreparation of 3'-and 5'-Labeled RNAs. Viral RNA (vRNA) was extracted from a suspension of purified virus (13). The RNA was labeled at its 3' end as described (14) in a reaction containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesu...

The Genomes of Rotaviruses Isolated from Chronically Infected Immunodeficient Children

“…DI particles of other viruses, artificially created in the laboratory by passaging at high multiplicity of infection, contain shortened genomes, in contrast to the observation presented here. In the case of viruses possessing segmented RNA genomes the generation of DI particles is associated with the loss of normal large RNA segments and the appearance of small RNAs (DI RNAs), mostly migrating faster than the smallest normal RNA segment on polyacrylamide gels (Davis et al, 1980;Brown et al, 1983). The DI RNAs are generated either by internal or terminal deletions from the larger RNA (Davis et al, 1980;Holland et al, 1980) or by the formation of mosaic structures containing sequences from more than one of the larger RNA segments (Fields & Winter, 1982).…”

Genome packaging in influenza A virus

“…Naturally occurring DI RNAs are predominantly derived from the three largest segments (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998;Jennings et al, 1983). Examples from every segment have been identified, but as yet, no sequence data have been reported for a segment 7 DI RNA (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Liu & Air, 1993;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995).…”

“…Examples from every segment have been identified, but as yet, no sequence data have been reported for a segment 7 DI RNA (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Liu & Air, 1993;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995). The majority of DI RNAs studied have a single major internal deletion (Jennings et al, 1983;Nayak et al, 1982;Winter et al, 1981) and retain the terminal UTRs of their parent segment along with a variable amount of coding sequence (Davis et al, 1980;Davis & Nayak, 1979;Moss & Brownlee, 1981;Nakajima et al, 1979). The mechanism of DI RNA generation is not known, but it is likely that the size and location of deletions is determined by several factors, including the juxtaposition of non-contiguous sequences brought about by the coiled path of the vRNA in the RNP (Jennings et al, 1983).…”

“…Viruses containing DI RNAs arise in influenza A during high-multiplicity passage (von Magnus, 1954), particularly in certain genetic backgrounds (Nakajima et al, 1979;Odagiri & Tobita, 1990;Ueda et al, 1980). Sequencing of DI RNAs showed that they were derived from genomic segments by internal deletion, and retained the terminal regions of the segment (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Moss & Brownlee, 1981;Nakajima et al, 1979;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995). As it became possible to determine the segment from which a DI RNA was derived, it became apparent that in many cases, their presence correlated with reduced amounts of the parent segment in virus particles (Akkina et al, 1984;Nakajima et al, 1979;Odagiri & Tobita, 1990;Ueda et al, 1980).…”

“…This segment-specific competition implied that packaging involved selection for distinctive features shared by the segment and its DI RNA. The terminal regions retained in DI RNAs included portions of the coding region as well as the UTRs (Davis et al, 1980;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Liu & Air, 1993;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995) (see also Fig. 4).…”