Point substitution in a promoter-like region and the V1 gene affect the host range and symptoms of maize streak virus.

The nucleotide sequences of full-length infectious clones of two symptomatic and host range variants (MSV-Ns and MSV-Nm) of the Nigerian strain of maize streak virus (MSV) have been determined and shown to differ by only three nucleotides. MSV-Ns produced symptoms in infected maize plants sooner and the streaks were wider and more chlorotic than those of MSV-Nm; variant MSV-Ns also had a wider host range within the Gramineae. None of the three nucleotide differences resulted in amino acid changes. Site-directed mutagenesis showed that a substitution at nucleotide (nt) 40 in the V1 gene affected streak width, while severity of chlorosis, length of streaks, latency, and host range was determined by a single base change at nt 2473 in the large intergenic region. The nt 2473 change altered a potential promoter sequence (TATA box) in MSV-Ns 101 nucleotides upstream of the initiation codon of the C1 gene. Mutagenesis of TATA sequences located downstream of TATA -101 showed that TATA -101 alone was sufficient to confer a wide host range phenotype on MSV-Ns and suggested that it might function as a promoter for the expression of complementary-sense open reading frames. When compared with an updated promoter consensus derived from genes of the Gramineae, the promoter context around TATA -101 in MSV-Ns was not more favorable than those found at -57 and -62 in MSV-Nm.

Host range and symptoms are determined by specific domains of the maize streak virus genome.

We have cloned two distinct symptomatic variants of the geminivirus streak virus from maize plants infected with the Nigerian strain (MSV-N). Following "agroinoculation" to maize plants MSV-Nm produces narrow, mildly chlorotic discontinuous streaks, whereas MSV-Ns-infected tissue has wide, severely chlorotic streaks. Symptom appearance is delayed following MSV-Nm inoculation. MSV-Nm has a narrow host range within the Gramineae comprising a fraction of that of the wide host range isolate MSV-Ns. The two isolates are highly homologous and have identical restriction enzyme maps. In order to localize the determinants of pathogenicity we constructed, in vitro, hybrid genomes by restriction enzyme fragment exchange. The determinants of host range, severity of chlorosis, streak length, and timing of symptom appearance map to a fragment which includes the large intergenic region and the 5' terminus of the complementary sense C1 gene. Streak width is determined by the virion-sense portion of the genome, which is consistent with the observation that the virion-sense gene products (V1 and V2) are required for spread of the virus.

Mutational analysis of the virion-sense genes of maize streak virus.

Insertion and deletion mutagenesis of the two virion-sense genes, V1 and V2, of maize streak virus (MSV) prevents symptomatic infections following Agrobacterium-mediated 'agroinoculation' of maize seedlings. These genes code for an Mr 10900 protein and for coat protein, respectively. Mutants containing insertions or deletions in the coat protein gene, V2, were able to replicate to low levels, producing dsDNA although virion ssDNA was not detected and symptoms were not observed. Hence, unlike the bipartite geminiviruses, MSV requires coat protein to produce symptomatic systemic infection. Mutations in gene V1 which considerably shortened the Mr 10900 protein (V1 gene) resulted either in low levels of replication, in which all the DNA forms associated with a wild-type infection were produced, or in no infection, in which case coat protein production may also have been affected. A V1 mutant generated in vivo with 11 of the 14 N-terminal amino acids altered, was viable and produced symptoms typical of a wild-type infection. Infectivity, assessed by replication and symptom expression, was restored by co-inoculating constructs containing single mutations in different open reading frames, thus rescue can occur by trans-complementation of gene products. The experiments showed that the mutations did not affect the nucleotide sequence requirements for replication and that in all cases intermolecular recombination eventually resulted in dominant wild-type virus.

Pea early browning virus RNA1 encodes four polypeptides including a putative zinc-finger protein.

We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.