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Rice dwarf phytoreovirus (RDV) is an icosahedral double-shelled particle approximately 70 nm in diameter (2, 5). The core particle, which is composed of 12 segments of double-stranded RNA and four different proteins, is enclosed by a capsid that consists of proteins designated P2 and P8 (18). RDV is not transmissible mechanically but is transmitted to rice exclusively by the leafhopper Nephotettix cincticeps and some other leafhopper species. RDV proliferates within the leafhopper vectors. An unusual biological characteristic of this virus is its ability to multiply in both plants and certain invertebrates. Investigations of the molecular features of the virus that allow it to proliferate in members of both the plant and animal kingdoms are of great interest from a biological perspective, as well as from a practical perspective, since such investigations should help us to find a way to interrupt the cycle of viral infection.

The extent of infection by the virus of vector cells in monolayer cultures (VCM) was reduced with reductions in the amount of P2 protein in the transmission-competent (TC) isolates of RDV (TC-RDV) that were used for inoculation (18). Particles from which P2 had been specifically removed by treatment with CCl₄ failed to proliferate in insect vectors by membrane feeding, a result that suggests that P2 might be required for infection of insect vectors by the virus. Furthermore, a transmission-defective (TD) isolate of RDV (TD-RDV) which was unable to infect VCM lacked P2, probably as the result of a point mutation in the open reading frame that encodes the protein (17). All these results indicate that the P2 protein is one of the factors that is essential for infection of vector cells by the virus and, thus, that the P2 protein influences transmissibility by insect vectors. In the present study, we examined in further detail the function of the P2 protein during the infection of cells of the insect vector.

The O strain of RDV (7) has been maintained for 16 years in rice plants by sequential inoculations, with N. cincticeps as the vector, at least once a year. For the present study, the virus was purified from infected rice plants with CCl₄ (11) or without CCl₄ (18). Infected rice leaves were macerated in a meat chopper, and the slurry of chopped leaves was subjected to differential centrifugation and to consecutive sucrose density gradient centrifugations on 10 to 40 and 40 to 60% sucrose. The final pellet, after high-speed centrifugation of the purified virus, was suspended in a 0.1 M solution of histidine that contained 0.01 M MgCl₂, pH 6.2 (His-Mg). TC-RDV purified without CCl₄ included both P2 and P8 as outer-capsid proteins (intact TC-RDV), while TC-RDV which was purified with CCl₄ lacked the P2 protein (P2-free TC-RDV) (18). A TD isolate purified without CCl₄ from infected plants vegetatively propagated for 12 years in a greenhouse lacked the P2 protein (P2-free TD-RDV) (17).

To determine whether the failure of particles that lacked P2 to infect VCM was due to the inability of the particles to interact with the insect cells, we examined the ability of viral particles to adsorb to VCM. The cell line NC-24, originally established from embryonic fragments dissected from N. cincticeps eggs (8), has been maintained for 19 years by subculturing at intervals of 7 to 10 days. We tested the VCM for the presence of viral antigen after incubation of the cells in intimate contact with viral particles. Three milliliters of medium containing 500 µg of purified virus was overlaid on VCM that had been cultured for 7 days in 40-ml flasks with a 25-cm² flat bottom (NUNC, Roskilde, Denmark). After a 2-h incubation at 25°C, the inoculum was removed and the VCM were washed three times with His-Mg. The cells were harvested after addition of 0.05% trypsin (6) and were resuspended in 500 µl of His-Mg and stored at 70°C. After thawing, the cells were macerated with a Teflon homogenizer. Each homogenate was centrifuged for 5 min at 2,500 × g, and the supernatant was subjected to serial twofold dilutions for analysis by an enzyme-linked immunosorbent assay that was designed to reveal the presence of viral antigen (16). As shown in Fig. 1, high titers of viral antigen were detected in cells that had been treated with intact TC-RDV. By contrast, no antigen was detected in cells after treatment with P2-free TC-RDV or with P2-free TD-RDV. These results, obtained with different materials, suggest that P2 plays an important role in the interaction of RDV with VCM. The adsorption of intact RDV to VCM seemed to be specific, in view of the fact that intact TC-RDV, as well as P2-free TC-RDV and TD-RDV, failed to adsorb to High Five cells that originated from the nonhost insect Trichoplusia ni (Invitrogen, San Diego, Calif.). These results, together with the observation that P2-free TC-RDV and TD-RDV did not interfere with infection by intact TC-RDV (data not shown), suggest that P2 protein plays a role in the attachment of virus particles to cells of the insect vector.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Adsorption of RDV particles to insect cells, as indicated by the reciprocals of dilution endpoints that gave positive results in an enzyme-linked immunosorbent assay. Results are shown for VCM that had been incubated with intact RDV (bar 1), VCM incubated with P2-free TC-RDV (bar 2), VCM incubated with P2-free TD-RDV (bar 3), High Five cells incubated with intact RDV (bar 4), and VCM incubated with His-Mg buffer (bar 5).

To confirm that viral particles had penetrated the vector cells, we examined thin sections of VCM that had been exposed to intact TC-RDV by electron microscopy. VCM on a coverslip (15 mm in diameter) were covered with 0.1 ml of medium that contained approximately 20 µg of intact TC-RDV or P2-free TC-RDV. After being incubated for 2 h at 25°C and washed with His-Mg as described previously (13), the cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 2 h at 0°C. They were postfixed in 2% osmium tetroxide for 3 h at 6°C, dehydrated in a graded acetone series, and embedded in Spurr's low-viscosity epoxy resin (14). After the resin hardened, the VCM were detached from the coverslip, mounted on a resin block, and sectioned. The sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (H-7000; Hitachi, Hitachinaka, Japan). Characteristic intact virus particles of the expected size were observed on the surface of all cell membranes and in the vesicles of monolayer cells that had been exposed to intact TC-RDV (Fig. 2A). No such particles were found in VCM which had been incubated with P2-free TC-RDV (Fig. 2B). The results demonstrated that RDV particles that penetrated the VCM were in the double-layered form. This conclusion was further supported by electron microscopic observations of dipped preparations, stained with uranyl acetate, of the materials that had been used for enzyme-linked immunosorbent assay. The particles were double layered and appeared intact (12), and they reacted with an antiserum against P8 outer-capsid protein purified by the method of Zhu et al. (19) as described by Milne and Luisoni (9) (data not shown).

View larger version (116K): [in this window] [in a new window]   FIG. 2.   Electron microscopy of thin sections of VCM derived from the insect vector N. cincticeps after incubation with intact (A) or P2-free (B) TC-RDV. Bars, 200 nm.

The inability of particles that lacked P2 to infect VCM (18) appeared to be due to their inability to attach to the insect cells (Fig. 1) rather than to failure in initiating infection after the entry of virus particles into the cells. The same phenomenon might also occur at the surfaces of cells in the intestinal tracts of insects that are allowed to feed on virus particles through a membrane. Thus, we next examined the possibility that P2-free particles might be infectious if they were introduced into the insect by injection into the abdomen. The method for injection of virus particles into nymphs has been described elsewhere (11). Insects that had been injected with virus particles or that had been allowed to feed on particles through a membrane were reared on healthy rice seedlings for 18 days. They were then confined individually for 2 days to test tubes that contained rice seedlings and allowed to feed on and, thus, to inoculate the rice seedlings. Each insect was then examined individually by enzyme-linked immunosorbent assay for the presence of the viral antigen, as described elsewhere (16). The inoculated rice seedlings were grown in an air-conditioned greenhouse (27 ± 3°C). The seedlings were examined for symptoms of RDV infection 40 days after inoculation.

As shown in Table 1, injected P2-free TC-RDV, as well as injected intact TC-RDV, was able to infect and proliferate in insects, as determined by enzyme-linked immunosorbent assays of individual insects. These insects were also able to transmit the virus to rice seedlings, as judged by the appearance of symptoms on rice plants. By contrast, in membrane-feeding tests, only intact TC-RDV was able to infect insects, which were then able to transmit the virus to rice seedlings (18). These results demonstrate that P2-free TC-RDV retained infectivity when introduced into insect bodies. On the other hand, P2-free TD-RDV did not proliferate in intact insects nor was it transmitted to plants by either the membrane-feeding or the injection method, suggesting that P2 has additional functions.

The glass capillaries used for injections allowed particles that lacked P2 to infect and multiply in the insects. This result implies that P2-free particles cannot enter cells of the intestinal tract, where virus particles are thought to infect the vector (1), but can infect and multiply once they have been introduced into the hemolymph, bypassing the wall of the gut, which forms a barrier to infection. This hypothesis is supported by earlier results which showed that insects of nontransmitting races of the leafhopper Cicadulina mbila became infective when fed on maize streak virus-infected plants and punctured with a fine needle to allow materials in the digestive system to be introduced into the insects' bodies (15). Clearly, there are cells in the insect vector that are sensitive to RDV particles that lack P2. That is, once P2-free TC-RDV has been introduced physically into the hemolymph and then to hypothetical sensitive cells, or directly into cells through openings in cell membranes, infection can occur in the absence of P2. After the onset of the initial infection, all the viral proteins, including P2, can be expressed and the normal multiplication cycle can occur. The RDV produced in cells that have been initially infected with P2-free TC-RDV can infect neighboring cells, as can intact TC-RDV. When RDV was purified from plants infected by insects that had been injected with P2-free TC-RDV, the virus was found to contain P2 and other protein components at levels similar to those of the corresponding components of native RDV (data not shown). These results, together with the observation that P2-free particles did not make intimate contact with insect cells and were unable to enter them (Fig. 1), demonstrate that the P2 protein of RDV allows virus particles to adsorb to cells of the insect vector. Thus, P2 seems to be one of the molecules that is essential for infection of insect cells by RDV.

Among the structural proteins of viruses belonging to the family Reoviridae, 1 of reovirus (10) and VP4 of rotavirus (3, 4) correspond to RDV P2 in their outer capsid location in virus particles and in their functions in attachment to host cells (thus being associated with viral infectivity), although no significant homology was found between these proteins and P2. Detailed comparative study of the structure-function relationships of these proteins may provide insights into the mechanism that determines the host specificities of the viruses.