The nature of the amino acids in the N-terminal ‘DAGX’ motif of the coat protein of tobacco vein mottling virus (TVMV) that have a direct effect on aphid transmissibility of the virion were further defined by sitedirected mutagenesis. In the first position of the DAGX motif, Asp or Asn are required for aphid transmissibility. In the second position, the nonpolar residue Ala, but not the nonpolar Gly or Val or the polar Thr and Ser, is compatible with transmissibility. In the third position, the small, neutral, nonpolar Gly appears to be critical; even substitution of Ala, with a minimal side-chain, drastically reduces transmissibility. Although the amino acid following the DAG sequence is not highly conserved among potyviruses, the presence of an acidic Glu or Asp residue at this position in the TVMV coat protein drastically reduces or abolishes aphid transmissibility. An attempt was made to test the hypothesis that trypsin cleavage of the N terminus is involved in the aphid inoculation process by destroying a trypsin cleavage site downstream from the DAGX motif. While the predicted decrease in transmission occurred from infected plants, there was no effect on the transmission of purified virus. Of the 23 mutations in the DAGX region of TVMV reported here and previously, only two, substitutions of Lys and Arg for Asp, had a detectable adverse effect other than on aphid transmissibility. These, and perhaps other, residues near the N terminus function in some phase of the TVMV life cycle, in addition to aphid transmission.
AtreyaC. D.,
AtreyaP. L.,
PironeT. P.1990b; A strategy for rapid identification and selection of site-directed low frequency point mutations. BioTechniques 9:702–703
AtreyaC. D.,
AtreyaP. L.,
PironeT. P.1990c; Selection of deletion mutants by polymerase chain reaction. Biochemical and Biophysical Research Communications 173:1344–1346
AtreyaP. L.,
AtreyaC. D.,
PironeT. P.1991; Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences, USA 88:7887–7891
BoyeK.,
JensenP. E.,
SlummannB. M.,
HenningsenK. W.1990; Nucleotide sequence of cDNA encoding the BYMV coat protein gene. Nucleic Acids Research 17:4926
DoljaV. V.,
HaldemanR.,
RobertsonN. L.,
DoughertyW. G.,
CarringtonJ. C.1994; Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO Journal 13:1482–1491
DomierL. L.,
FranklinK. M.,
ShahabuddinM.,
HellmannG. M.,
OvermeyerJ. FI.,
HiremathS. T.,
SiawM. F. E.,
LomonossoffG. P.,
ShawJ. G.,
RhoadsR. E.1986; The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Research 14:5417–5430
DomierL. L.,
FranklinK. M.,
HuntA. G.,
RhoadsR. E.,
ShawJ. G.1989; Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proceedings of the National Academy of Sciences, USA 86:3509–3513
HammondJ.,
HammondR. W.1989; Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene. Journal of General Virology 70:1961–1974
HarrisonB. D.,
RobinsonD. J.1988; Molecular variation in vector-borne plant viruses: epidemiological significance. Philosophical Transactions of the Royal Society of London B321:447–462
KunkelT. A.,
RobertsJ. D.,
ZakourR. A.1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
ShuklaD. D.,
InglisA. S.,
McKernN. M.,
GoughK. H.1986; Coat protein of potyviruses. 2. Amino acid sequence of the coat protein of potato virus Y. Virology 152:118–125
ShuklaD. D.,
ThomasJ. E.,
McKernN. M.,
TracyS. L.,
WardC. W.1988; Coat protein of potyviruses. 4. Comparison of biological properties, serological relationships, and coat protein amino acid sequences of four strains of potato virus Y. Archives of Virology 102:207–219
UyedaI.,
TakahashiT.,
ShikataE.1991; Relatedness of the nucleotide sequence of the 3′-terminal region of clover yellow vein potyvirus RNA to bean yellow mosaic potyvirus RNA. Intervirology 32:234–245