We analyzed the distribution of the caulif lower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF-and the derivative mutant-microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunof luorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.Cauliflower mosaic virus (CaMV) was the first DNA plant virus to be reported (1); it is the type member of the genus Caulimovirus and is characterized by icosahedral particles 50 nm in diameter. The complete genome is a double-stranded circular DNA of Ϸ8000 bp encoding at least six genes which are believed to be independently translated from a full genome length polycystronic 35S RNA (for a review, see ref.2).CaMV is transmitted between host plants by several aphid species in a noncirculative manner (ref. 3; for a review about plant virus vector transmission, see refs. 4 and 5). After acquisition from an infected plant, the virus does not circulate within the vector's body and is retained for a relatively short time (few hours), probably on the cuticular lining of the aphid feeding apparatus. During subsequent feeding on a healthy plant, the virus is released to initiate a new infection. Lung and Pirone (6, 7) demonstrated that aphid transmission is regulated by an aphid transmission factor (ATF) that has been identified as the expression product of CaMV gene II (8-11). The ATF is a nonstructural polypeptide of 18 kDa (P18) and appears to have no other function in the CaMV life cycle. Indeed, isolate CM4-184, a naturally occurring mutant which completely lacks gene II, is infectious in host plants but is not aphid transmissible (12).Gene II of an aphid-transmissible CaMV isolate has been expressed to produce P18 in Sf9 insect cells via a baculovirus recombinant (13), which is biologically active (14). P18 and the virus can be acquired separately; aphids that are first fed through a parafilm membrane with baculovirus expressed P18 become capable of transmitting the naturally non-aphidtransmissible CM4-184 isolate. Native P18 accumulates, both in baculovirus recombinant-infected Sf9 cells ...