RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus.
The 63 kDa '63K' movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA-protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.
Kalinina
N O
NO
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Rakitina
D A
DA
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Yelina
N E
NE
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Zamyatnin
A A
AA
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Stroganova
T A
TA
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Klinov
D V
DV
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, 117997 Moscow, Russia2.
Prokhorov
V V
VV
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, 117997 Moscow, Russia2.
Ustinova
S V
SV
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, 117997 Moscow, Russia2.
Chernov
B K
BK
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova St, Moscow 117984, Russia3.
Schiemann
J
J
Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany4.
Solovyev
A G
AG
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
Morozov
S Yu
SY
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia1.
D000595:Amino Acid Sequence; D001665:Binding Sites; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D008970:Molecular Weight; D053830:Plant Viral Movement Proteins; D010942:Plant Viruses / Q000235:genetics*; D012313:RNA / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D014764:Viral Proteins; D012313:RNA