A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission

Abstract

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surfaceoriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.Members of the family Luteoviridae are icosahedral viruses with small (Ϸ6-kb) RNA genomes that infect phloem-associated tissues of their plant hosts (29). They are transmitted between plants by aphids in a circulative, persistent manner. Although the virus can survive for extended periods in often hostile environments in the aphid, the virus does not replicate in the insect (11). Features of the virion regulate local and systemic movement of the virus in plant hosts and regulate the recognition, transport, and persistence of virus in aphid tissues, yet little is known about the specific properties and biologically active domains of the luteovirus particle.The icosahedral virions of members of the Luteoviridae are composed of two structural proteins, the major 22-to 24-kDa capsid protein (CP) encoded by open reading frame (ORF) 3, and a minor species referred to as the readthrough protein (RT) (17). The RT, encoded by ORF 3 and the downstream adjacent ORF 5, is translated by occasional suppression of the CP termination codon (16). The virion structure has not been resolved for any member of the Luteoviridae, but the capsids are thought to be assembled from 180 subunits according to T ϭ 3 quasi-symmetry. A variable but minor number of the 180 capsids are RT subunits incorporated into the virion via their CP moiety (16), although sequences in the RT domain regulate the incorporation (24). The Ϸ50-kDa RT domain encoded by ORF 5 is believed to protrude from the surface of the virio...

Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation

“…The results indicated that regardless of whether the RTD mutant incorporated RTP into the virion (RFI, EDE and SST) or not (QSS, GHPE, ERD and SYG), none of the RTD mutants were aphid transmissible (Table 2). Incorporated RTP, as well as surface loops of the CP, have been shown to be required for aphid transmission of poleroviruses (Brault et al, 2003;Bruyere et al, 1997;Jolly & Mayo, 1994;Kaplan et al, 2007;Lee et al, 2005). The results reported here showing that non-incorporating RTP mutants were not aphid transmissible are consistent with previous reports that RTP is required for aphid transmission.…”

“…Using Agrobacterium tumefaciens cultures containing the fulllength cDNA of the PLRV WT or one of the mutant viruses, agroinfiltration into mesophyll cells and agroinjection into vascular cells were done as described previously by Lee et al (2005) and Kaplan et al (2007). Translation and accumulation of RTP was measured in agroinfiltrated tissue samples by Western blot analysis as described previously in Lee et al (2005) and Kaplan et al (2007). Nicotiana benthamiana, N. clevelandii, Physalis floridana and Solanum sarrachoides plants were agroinjected and analysed for systemic infection as described previously by Lee et al (2002) and Kaplan et al (2007).…”

“…Systemically infected P. floridana or N. clevelandii plants were used as virus source tissue in aphid transmission assays. The tests were performed as described previously by Lee et al (2005) with the exception that five aphids were placed onto each plant. A 48 h acquisition access period was followed by a 4-5 day inoculation access period.…”

“…The P17 movement protein was not required for infection in the Nicotiana species, but it was required for infection in P. floridana and Solanum tuberosum (Lee et al, 2002). Virion formation is essential for virus movement in the Nicotiana species, P. floridana and S. tuberosum, but mutations in the CP demonstrated that infection efficiencies varied among host plants (Kaplan et al, 2007;Lee et al, 2005).…”

“…Multiple hosts were tested because host-specific effects have been reported for mutations in both the PLRV 17 kDa movement protein (P17) (Lee et al, 2002) and CP (Kaplan et al, 2007;Lee et al, 2005). The P17 movement protein was not required for infection in the Nicotiana species, but it was required for infection in P. floridana and Solanum tuberosum (Lee et al, 2002).…”