Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereas GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.Vaccinia virus (VACV), a member of the family Poxviridae, undergoes an elaborate assembly process involving the sequential formation of morphologically distinct infectious virus particles (6). The mature virions (MVs) are the most abundant infectious particles and are released upon cell lysis. The MV consists of (i) a dumbbell-shaped core containing the viral double-stranded DNA genome, structural proteins, and enzymes and factors for early gene transcription; (ii) lateral bodies of undefined nature that are nestled in the concavities of the core; and (iii) an external lipoprotein membrane. Membranes derived from either the trans-Golgi network or endosomal cisternae envelop a subset of MV particles to form doubly wrapped virions (WVs) (11,27,31). WVs traffic along microtubules to the periphery of the cell, where the outer membrane fuses with the plasma membrane to release enveloped virions (EVs) (12,23,36,37). Many EVs remain associated with the outer surface of the plasma membrane and mediate spread to neighboring cells on the tips of actin-containing microvilli (2, 30). Some poxviruses (e.g., cowpox, ectromelia, raccoonpox, fowlpox, and canarypox viruses, but not VACV or variola virus) occlude MVs in a proteinaceous body called an A-type inclusion (ATI) that is thought to protect the virus particles under harsh environmental conditions (17).Recent mass spectroscopy studies indicate that MVs are comprised of approximately 80 polypeptides (5, 22, 39). Al...

Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.