No abstract

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.The interaction between virus and plant proteins determines the capability of the virus to multiply and systemically infect the host plant (1). Systemic infection with plant viruses requires cell-to-cell and long-distance movement of viral genomic RNA (2, 3). Many plant viruses find access into cells through wounds. Upon initial entry into a plant cell, the virus multiplies and moves locally from cell to cell (local infection). In most cases, the transfer of viral RNA between cells is supported by a virus-encoded movement protein(s) (4-7), whereas in some viruses the capsid protein is a primary determinant of cell-to-cell movement (8-10). The movement protein interacts with the plasmodesmata and transfers the viral RNA into a neighboring uninfected cell (11-13). During systemic infection, the virus moves through the vascular system and multiplies in upper uninoculated leaves. Movement into the upper uninoculated tissue is a critical step in the infection by many plant viruses, and prevention of this step can result in significant protection of the host(s) from virus invasion (14). Coat proteins (CPs) of several plant viruses are essential for long-distance movement (15-23). Moreover, some RNA plant viruses, such as tobacco mosaic virus (TMV) (16,17,24), cowpea mosaic virus (25), alfalfa mosaic virus (AlMV) (18), tobacco etch potyvirus (26), and red clover necrotic mosaic virus (27) require functional CP for long-distance movement.Here, to study the role of AlMV CP in long distance movement, we designed a hybrid virus consisting of TMV, the type member of the tobamovirus group, and the CP of AlMV. The TMV genome consists of a single plus-sense RNA (6,395 nt) encapsidated with a 17.5-kDa CP, which results in rodshaped particles (300 nm in length). In addition to CP, TMV has three nonstructural proteins. Proteins (183 and 126 kDa) are translated from genomic RNA and are required for virus replication (28). The 30-kDa protein is a movement protein and pro...

Human respiratory syncytial virus (RSV) is the primary cause of respiratory infection in infants worldwide. Currently there is no available vaccine, although studies in animal models have demonstrated protective immunity induced by an epitope of the RSV G-protein representing amino acids 174-187. Two peptides containing amino acids 174-187 of the G-protein of the human RSV A2 strain (NF1-RSV/172-187 and NF2-RSV/170-191) were separately engineered as translational fusions with the alfalfa mosaic virus coat protein and individually expressed in Nicotiana tabacum cv. Samsun NN plants through virus infection. RSV G-protein peptides were expressed in infected plant tissues at significant levels within 2 wk of inoculation and purified as part of recombinant alfalfa mosaic virions. BALB/c mice immunized intraperitoneally with three doses of the purified recombinant viruses showed high levels of serum antibody specific for RSV G-protein and were protected against infection with RSV Long strain.

The Hepatitis Delta Virus (HDV) ribozyme self-cleaving activity in 20 M formamide solutions is unique. Does this catalytic activity result from the conservation of its tertiary structure in 20 M formamide? We followed the ribozyme structure in formamide solutions by monitoring the amount of bound Ethidium Bromide (EB). We were able to measure the quantity of dye bound using time-resolved fluorescence spectroscopy, as an estimate of the ribozyme double helical content. This method, calibrated by using oligonucleotides with defined tertiary structure and denaturing solvents, parallels NMR and UV measurements as a function of temperature. Measurements with the HDV ribozyme lead to three conclusions: (a) both the precursor and product RNAs are structured to 24 M (95% w/w) formamide or 4 M H2O solutions which is equivalent to 4 M H2O; (b) the HDV ribozyme is the only RNA sequence investigated in this study that retains so much structure in formamide; and (c) DNA analogs of formamide resistant HDV ribozyme sequences lose their structure at less than 15 M formamide. Thus, the structural integrity of the HDV ribozyme is an intrinsic property of the RNA molecule and its sequence.