Deciphering the Mechanism of Defective Interfering RNA (DI RNA) Biogenesis Reveals That a Viral Protein and the DI RNA Act Antagonistically in Virus Infection

Abstract

Virus accumulation was reduced when the 8K ORF was disrupted but D RNA was produced. Conversely, the virus accumulated at higher titers when the 8K ORF was intact and D RNA production was blocked. These data demonstrate that the D RNA interferes with virus infection and therefore should be referred to as a defective interfering RNA (DI RNA). The 8K protein was shown to be a weak silencing suppressor. This study provides an example of the interplay between a pathogen and its molecular parasite where virus accumulation was differentially regulated by the 8K protein and DI RNA, indicating that they play antagonistic roles and suggesting a mechanism by which the virus can attenuate replication, decreasing viral load and thereby enhancing its efficiency as a parasite.T he small genome size of RNA viruses means that most viral proteins are multifunctional and that viruses adopt several different strategies for gene expression including overlapping open reading frames (ORFs). In the course of infection many RNA viruses produce defective RNAs (D RNAs), which are deleted versions of the viral genome (see references 1 and 2 for a review). Because they lack essential genetic information, in plants D RNAs are totally dependent on the infectious helper virus for replication, encapsidation, and cell-to-cell and long-distance movement. Interference occurs when D RNA reduces symptom production by the parent virus or/and reduces multiplication of the helper virus; hence, the term defective interfering RNA (DI RNA) is used.Production of D RNA molecules during replication cycles seems to be a common occurrence in many viruses. D and DI RNAs of plant viruses, including DNA and negative-and positivestranded RNA viruses, have been described and characterized previously (1, 2). In some instances, for example, the criniviruses, DI RNAs consist of a mosaic of the multipartite parental viral genome (3). Multiple-deletion DI RNAs are typical for several members of genus Tombusvirus, and these DI RNAs have been extensively studied (4, 5). On the other hand, single-deletion D and DI RNAs have been reported in Potexvirus and Tobravirus genera as well as in several soilborne viruses of the genera Furo-, and Benyvirus (1).Although the DI RNA could represent up to 60% of virusspecific RNA in the infected plants, as in case of tombusviruses, the percentage of encapsidated DI RNAs could be as low as 3 to 4%, and most DI RNAs are not transmitted by vectors (1, 2, 6). Moreover, while most DI RNAs can efficiently accumulate in inoculated tissue, they do not always move systemically, as exemplified by cucumber mosaic virus (CMV) D RNA, which moves long distances in tobacco species but not in tomato, zucchini squash, or muskmelon (1,7,8).Three major mechanisms of interference by DI RNAs have been recognized (reviewed in references 2 and 5). These mechanisms are the following: (i) competition for viral and host resources, which impairs virus replication and attenuates the symptoms; (ii) DI RNA-triggered gene-silencing response; (iii) modulation of the...

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