?(Fig.4A).4A). filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar. Cytoskeletal elements have previously been demonstrated to be involved in several aspects of virus assembly (39, 66). For example, vaccinia virus has been shown to associate with actin during its release from the plasma membrane (15), while adenovirus is transported through the cytoplasm to the nucleus through its interaction with microtubules (17, 38). Actin has been implicated in the transport of baculovirus nucleocapsids to the nucleus (10). Other viruses contain actin in their envelopes along with viral surface glycoproteins, implying some role in the budding process (34, 54, 58). In addition, cytochalasin D, a disruptor of microfilaments, has been shown to impair the assembly of a number of different viruses (18, 42, 45). Most viruses use preexisting microtubule or microfilament proteins derived from host cells in these processes. However, we have recently demonstrated that insect poxviruses establish their own filament network during the later stages of infection, using a protein encoded by the viral genome (2). Entomopoxviruses (EPVs) are insect pathogens which replicate in the cytoplasm of infected cells and are members of the poxvirus family (reviewed in references 3 to 5 5 and 22). The genomes of these viruses consist of linear double-stranded DNA molecules which L189 are 130 to 300 kb in length. EPV (AmEPV) can be grown in cultured insect cells and is the most studied member of this group of viruses (22C25, 27, 40, 50). AmEPV derives its name from the Indian red army worm, a larva from the Lepidoptera family and the host from which the virus was originally isolated (23, 25, 50). Baculoviruses also infect Lepidoptera larvae but instead replicate in the nuclei of their host cells (44). A number of baculoviruses have been studied, but knowledge of nuclear polyhedrosis virus (AcNPV), which infects a wide variety of larvae including that of the alfalfa leaf hopper, is most extensive (44). This virus is used routinely to produce recombinant proteins in insect virus expression systems (36, 44, 46, 49). A common property of EPVs and baculoviruses is the formation of large intracellular structures known as occlusion bodies which assemble during the late stages of viral infection. Virions are embedded within these occlusion bodies, and the process serves to protect the virus from the external environment. In the case of baculoviruses, the occlusion bodies L189 are called polyhedra and are composed predominantly of a 31-kDa protein called polyhedrin (52). The occlusion bodies of EPVs are known as spheroids and consist mainly of a 110-kDa protein known as spheroidin (6, 9, 27, 55). Spheroidin and polyhedrin do L189 not appear to exhibit sequence homology (6, 27, 52). A multilamellar envelope also appears to surround both polyhedra and spheroids and may help to stabilize these structures during assembly (2, 53). During the late phases of AmEPV and baculovirus infections, large bundles of filaments also appear to accumulate in the infected insect cells. In the case of AmEPV, these structures are present in the cytoplasm (2, 22, 23, 40), while those found in cells infected with Rabbit Polyclonal to PHLDA3 baculoviruses reside both in the cytoplasm and in the nucleus (1, 14, 57). Baculovirus fibrils are composed primarily of a 10-kDa protein called p10 (47, 59). The p10 gene sequences from AcNPV, nuclear polyhedrosis virus (OpNPV), nuclear polyhedrosis virus, nuclear polyhedrosis virus, nuclear polyhedrosis virus (SeNPV), and nuclear polyhedrosis virus (CfNPV) have been reported (13, 32, 35, 66C68). Although the different.