Cross-linking Measurements
of the <i>Potato leafroll
virus</i> Reveal Protein Interaction Topologies Required for
Virion Stability, Aphid Transmission, and Virus–Plant Interactions
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Abstract
Protein interactions are critical determinants of insect
transmission
for viruses in the family <i>Luteoviridae</i>. Two luteovirid
structural proteins, the capsid protein (CP) and the readthrough protein
(RTP), contain multiple functional domains that regulate virus transmission.
There is no structural information available for these economically
important viruses. We used Protein Interaction Reporter (PIR) technology,
a strategy that uses chemical cross-linking and high resolution mass
spectrometry, to discover topological features of the <i>Potato
leafroll virus</i> (PLRV) CP and RTP that are required for the
diverse biological functions of PLRV virions. Four cross-linked sites
were repeatedly detected, one linking CP monomers, two within the
RTP, and one linking the RTP and CP. Virus mutants with triple amino
acid deletions immediately adjacent to or encompassing the cross-linked
sites were defective in virion stability, RTP incorporation into the
capsid, and aphid transmission. Plants infected with a new, infectious
PLRV mutant lacking 26 amino acids encompassing a cross-linked site
in the RTP exhibited a delay in the appearance of systemic infection
symptoms. PIR technology provided the first structural insights into
luteoviruses which are crucially lacking and are involved in vector–virus
and plant–virus interactions. These are the first cross-linking
measurements on any infectious, insect-transmitted virus