Alfalfa mosaic virus temperature-sensitive mutants. V. The nucleotide sequence of TBTS 7 RNA 3 shows limited nucleotide changes and evidence for heterologous recombination.

Nucleotide sequence determination of the coat protein cistron of the alfalfa mosaic virus (AIMV) temperature-sensitive mutant, Tbts 7 (uv) revealed a small number of point mutations of which only one results in the replacement of an amino acid: the asparagine residue at position 126 is replaced by an aspartate residue. RNA transcribed in vitro from a Tbts 7 cDNA 4 clone directed the production in vitro of a polypeptide which shows the same altered electrophoretic mobility in SDS-polyacrylamide gels as the Tbts 7 coat protein. Nucleotide sequence analysis of the 32-kDa open reading frame revealed some base changes, but none of these lead to changes in the primary structure of the protein. The 5'-terminal sequence of Tbts 7 RNA 3 was analyzed by cDNA cloning. At least three different types of nontranslated leader sequences were found, indicating considerable heterogeneity at the 5' end of the mutant RNA 3. The results indicated that the low abundance of RNA 3-containing particles in Tbts 7 virus preparations might be due to malfunctioning of the 5' terminus of Tbts 7 RNA 3 during replication.

Nucleotide sequence of narcissus mosaic virus RNA.

The nucleotide sequence of the genomic RNA of narcissus mosaic virus (NMV) was deduced from a set of cDNA clones and by direct sequencing of RNA. The genome, with a length of 6955 nucleotides [excluding the 3'poly(A) tail], contains six open reading frames (ORFs) with the capacity to code for polypeptides of more than 10K, with Mr values of 186284, 25845, 13998, 11059, 26097 and 10519. The first five of these putative proteins show considerable homology to similar proteins encoded by the RNAs of potato virus X (PVX, 6435 nucleotides) and white clover mosaic virus (WClMV, 5845 nucleotides). The sixth ORF is completely overlapped by the Mr 26097 coat protein cistron and has some homology with a similar ORF in WClMV RNA. The difference in length of the RNAs of NMV, PVX and WClMV is due to a non-homologous domain of variable length in the central region of the 5'-proximal ORF of the three viruses.

The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses.

Double-stranded cDNA of potato virus X (PVX) genomic RNA has been cloned and sequenced. The sequence [6435 nucleotides excluding the poly(A) tract] revealed five open reading frames (ORFs) which were numbered one to five starting at the 5' terminus of the RNA. They encoded proteins of Mr 165588 (166K), 24622 (25K), 12324 (12K), 7595 (8K) and 25080 (coat protein), respectively. ORFs 1 and 2 were inphase coding regions. The ORF 1 product contained domains of homology with the tobacco mosaic virus 126K and 183K products. The ORF 2 and 3 products showed homologies with the barley stripe mosaic virus 58K and 14K proteins, the beet necrotic yellow vein virus 42K and 13K products and the white clover mosaic virus 26K and 13K products, respectively. The significance of these homologies with respect to putative functions of the PVX-encoded proteins are discussed.

Non-structural proteins and RNAs of alfalfa mosaic virus synthesized in tobacco and cowpea protoplasts.

Three proteins, reacting specifically with sera raised against synthetic peptides identical to C-terminal amino acid sequences in alfalfa mosaic virus (AIMV) proteins P1, P2, and P3 translated in vitro from the AIMV RNAs 1, 2, and 3, respectively, were for the first time observed in tobacco and cowpea protoplasts. Part of P2 is post-translationally modified in protoplasts, because the anti-P2 serum reacted also with a protein migrating slower than P2 itself. The modification reported for P3 in infected tobacco leaves (T. Godefroy-Colburn et al. (1986) J. Gen. Virol. 67, 2233-2241) was observed in AIMV-infected bean leaves but not in AIMV-infected protoplasts and is apparently not essential for viral replication. Time course experiments showed that all nonstructural proteins could be detected 6 hr postinoculation. The two largest proteins P1 and P2 disappeared when virus production had reached a plateau, while the smallest nonstructural protein P3 remained at a constant level. Cell fractionation experiments showed that minus-strand RNA as well as all viral-encoded proteins were found in the 1000 g subcellular fraction. This location differs from the location of the nonstructural proteins in infected tobacco leaves (A. Berna et al. (1986) J. Gen. Virol. 67, 1135-1147).

Alfalfa mosaic virus temperature-sensitive mutants. III. Mutants with a putative defect in cell-to-cell transport.

Forty-five mutants of alfalfa mosaic virus with a temperature-sensitive (ts) replication in tobacco leaf discs were assayed in cowpea protoplasts at 25 and 30 degrees . The extent of replication of 9 mutants at 30 degrees was less than 10% of that at 25 degrees, whereas the replication of 14 mutants was not affected at 30 degrees. Twenty-two mutants showed an intermediate ts behavior. Several mutants with a non-ts phenotype in cowpea protoplasts were also found to be non-ts in their replication in tobacco protoplasts. The observation that at 30 degrees these mutants are able to replicate in single cells but not in leaf discs suggests that they may be defective in a transportation function.

Human interferon does not protect cowpea plant cell protoplasts against infection with alfalfa mosaic virus.

Orchansky et al. (P. Orchansky, M. Rubenstein, and I. Sela), Proc. Natl. Acad. Sci. USA 79, 2278-2280, 1982) reported that natural and recombinant human interferon (HuIFN), e.g., HuIFN-beta and -alpha subspecies gamma3, protects plant cells from infection with a plant virus (tobacco mosaic virus). For their study they used tobacco leaf discs as well as tobacco protoplasts. It is now reported that recombinant HuIFN-beta and HuIFN-alpha2 do not significantly decrease alfalfa mosaic virus increase in cowpea mesophyll protoplasts.