Potato leafroll virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant viruses incorporated the RTP into the virion. None of the mutant viruses were aphid transmissible, but the viruses persisted in aphids for a period sufficient to allow for virus transmission. Several mutant viruses were examined further for systemic infection in four host species. All mutant viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

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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...

Bovine viral diarrhoea virus (BVDV) isolates infect cultured Madin-Darby bovine kidney (MDBK) cells as efficiently as sheep kidney cells. In contrast, border disease virus (BDV) propagates poorly in MDBK cells but infects sheep cells very efficiently. The envelope glycoprotein E2 has been shown to be essential for virus infectivity. To explore the potential role of E2 in pestivirus host range in cell cultures, we engineered a chimeric BVDV with the E2 coding region from BDV. As expected, the BVDV-E2 bdv chimera retained the ability of BDV to multiply in sheep cells but experienced a remarkable reduction in its ability to propagate and form plaques in MDBK, a phenotype that is characteristic of the E2 donor, BDV31 virus. Control chimeric BVDV bearing a type II E2 demonstrated that the heterologous E2 does not impair replication in MDBK or lamb cells. These results establish a role for E2 in determining the tropism of a pestivirus in cell culture.