A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.

In many tumor systems, the location and extent of metastases are nonrandom, suggesting that unique cell properties are important in tumor arrest and formation of secondary tumor colonies. The lung and the liver are among the most frequently involved organs. These organs are also the predominant site of metastases by tumor cells of a model system of chemically induced lymphomas in DBA/2 mice that we have been investigating (1-5). The system consists of a low-metastatic parental tumor (Eb), a spontaneous high-metastatic variant thereof (ESb), and a high-metastatic tumor line of independent origin (MDAY-D2).We previously described that ESb and MDAY-D2, but not Eb, tumor cells had the unique properties of adhesion to and invasion of normal lung tissue in vitro (1, 6). Now we describe another unique characteristic of the two metastatic tumor lines, namely, a selective binding to isolated hepatocytes in vitro.Recently Kolb et al. (7) demonstrated a lectin-like receptor on rat liver cells that has specificity for galactosyl residues and may play a role in the trapping of recirculating desialylated lymphocytes in the liver (8). In our study, we investigated whether a similar interaction might be responsible for the trapping of circulating metastatic tumor cells in the liver. Materials and MethodsTumor Cells. Eb is a Heidelberg subline of the methylcholanthrene-induced DBA/2 lymphoma L5178Y, and ESb is a spontaneous variant thereof with highly metastatic potential (1, 5). The etiology of the metastatic DBA/2 tumor MDAY-D2 has been described (9). The tumor cells were passaged in tissue culture using RPMI-1640 medium (Grand Island Biological Co., Grand Island, N. Y.) with 10% fetal calf serum (FCS) and 2 × 10 -~ M 2-mercaptoethanol, Murine Hepatocytes. Liver cell suspensions were prepared as previously described (10). An 8-12-wk-old mouse was injected with 0.3 ml of 3.6% chloral hydrate in phosphated-buffered saline and 0.1 ml of heparin (500 U/ml). The hepatic portal vein and inferior vena cava were cannulated with a 21-gauge needle and a 16-gauge plastic catheter, respectively. The liver was perfused in situ, first with perfusion buffer (10) that was saturated with 02 at 36°C for 2-3 min,

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane' revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the memtrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid, via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.

The purpose of this paper is to describe the immunocytochemical-localization of N and NS nucleocapsid proteins of vesicular stomatitis virus in the cells throughout the infectious cycle. N protein was detected in the cytoplasm at 2 h after infection and formed small cytoplasmic clusters which progressively increased in size and number. At 5-6 h, it formed large cytoplasmic inclusions. NS protein was detected in the cytoplasm a little later than N protein and showed almost the same immunostaining pattern. However, diffuse background staining of NS protein was identified throughout the cytoplasm by double immunostaining methods. At electron microscopic level, N protein was mostly granular and occasionally organized in strands at 2-3 h. At 5-6 h, numerous immunostained reaction products were organized in strands. The reaction products of NS protein were almost the same as those of N protein with the exception that diffuse background staining was observed. Cos cells, transfected with SV40 vector containing N gene obtained by recombinant DNA technique, showed clusters of N protein, but virtually no strand at electron microscopic levels. The rapid-freezing and deep-etching replica method demonstrated that loosely coiled VSV genome coated with N protein was localized on cytoplasmic sides of cell membranes in the infected cells. These results showed that complete virus genome replication was needed for strand formation of N and NS proteins and suggested that they were bound to VSV genomes in the infected cells.

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