The single-stranded DNA geminiviruses produce transcripts from both strands (virion- and complementary-sense) of a nuclear double-stranded DNA molecule. In maize streak virus (MSV)-infected maize plants, approximately 80% of the complementary-sense transcripts produce the C1 protein, whilst the remaining 20% are spliced to remove a 92 nt intron and produce a C1:C2 fusion protein (Rep). Disruption of the complementary-sense 3' splice site abolished virus replication. The majority of the virion-sense transcripts initiated one nucleotide upstream of the V1 (movement protein) gene and a minority a further 141 nucleotides upstream. A 76 nt intron, with features typical of plant introns, was identified within the V1 gene, upstream of the coat protein gene. Spliced and unspliced forms of each virion-sense transcript were produced, but they differed in splicing efficiency. Approximately 50% of the major transcript and less than 10% of the minor transcript were processed. Mutagenesis of the consensus 5' splice site in the V1 gene resulted in the use of alternative cryptic splice sites, confirming the importance of splicing for MSV infection. Spliced virion-sense transcripts were also identified in tissue infected with the closely-related Digitaria streak virus (DSV) but not with another subgroup I geminivirus, wheat dwarf virus. Collectively, the multiple transcript initiation sites and different splicing efficiencies suggest that splicing is an important feature in the regulation of both early and late gene expression in MSV and DSV.

Persistent infections were established in suspension cultures of BHK21/13S cells with both Parana and lymphocytic choriomeningitis viruses. Four generations after infection with either virus, more than 90% of the cells scored as infective centers, with concomitant peaks in extracellular virus yields. In both cultures the synthesis of detectable plaque-forming units (PFU) ceased about the 50th generation postinfection, and this condition was maintained until the 350th cell generation when the cultures were discontinued. The generation time of each culture was identical to that of uninfected parent controls, and at no time were cytopathic effects evident. In spite of the absence of infectivity, over 90% of the cells sampled at various times contained viral antigen demonstrable by immunofluorescence. When either of these persistently infected cell lines was substituted for normal cells in the standard plaque assay, very low efficiencies of plating were observed for homotypic and heterotypic viruses. Plaque formation by several heterologous viruses was virtually unaffected.

SUMMARYA Pichinde persistently infected BHK2I/t3S culture was established in which defective interfering (DI) virus continued to be synthesized after cessation of plaque-forming virus replication. This DI virus, concentrated from NaC1-polyethylene glycol treated tissue culture fluids, was shown to band over a much broader range than standard virus, in either discontinuous or continuous sucrose gradients. The polyacrylamide gel profile of the RNAs extracted from standard virus contained six components with sedimentation coefficients corresponding to 3 I, 28, 22, I8, 15 and 4-6S. All RNAs extracted from DI virus preparations, however, did not contain the 22 and I5 S species. Furthermore, a new 20 S fraction was observed in DI virus taken from cultures which had been maintained for more than I75 generations after the initial infection, whereas it was absent in DI virus synthesized prior to that time.

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