In recent years, the use of fluorescent molecules as protein tags, combined with advanced imaging techniques, has been instrumental in providing unprecedented opportunities for visualizing many dynamic processes in living cells (12
). By use of viruses genetically tagged with fluorescent proteins, several aspects of virus biology and virus-cell interactions in vitro and in vivo have been examined. In particular, fluorescent-tagged viruses were used to study intracellular transport of human immunodeficiency virus capsids (35
), capsid assembly (36
), and genome recombination (30
); herpes virus entry, capsid transport, and egress (33
); rhabdovirus gene expression (14
) and immune response (25
); and morbillivirus tissue tropism (53
). In this report, we generated a recombinant VSV encoding P protein fused in frame with eGFP (VSV-PeGFP), which allowed us to track the movement of viral nucleocapsids in infected cells by real-time imaging. Our results suggest that the nucleocapsids are synthesized at sites distributed throughout the cytoplasm and that the progeny nucleocapsids are transported to the cell periphery by an MT-mediated process. Our observation that nucleocapsids are in close proximity to mitochondria suggests that mitochondria may also be involved in the nucleocapsid transport process.
Insertion of eGFP into the hinge region of the P protein led to PeGFP fusion protein, which was active in viral genome transcription and replication, indicating that the hinge region can accommodate large insertions without much adverse effect on protein function. However, incorporation of PeGFP as well as the L protein into virions was affected (Fig. ). It is possible that the large size of the fusion protein might have sterically hindered the association of P-L complexes with the viral N RNA template, resulting in reduced incorporation of PeGFP and L proteins in the virions. Although the levels of viral macromolecular synthesis in cells infected with VSV, VSV-PeGFP, or VSV-GFP were not significantly different from each other, VSV-PeGFP grew to titers that were almost 10-fold less than those of the other viruses. Whether the reduced levels of incorporation of L and P proteins in VSV-PeGFP account for its retarded growth phenotype remains to be examined. We have recently demonstrated that the P protein of VSV plays a major role in assembly of infectious particles (7
). It is therefore possible that insertion of eGFP into P protein may have affected the assembly functions of the protein to some extent, resulting in retarded growth of VSV-PeGFP. It should be noted that a recombinant rabies virus encoding the P protein with eGFP fused in frame at its amino terminus possessed a significantly retarded growth phenotype (14
). In addition, eGFP incorporation into the L protein of measles virus (11
) led to a reduced growth phenotype of the recombinant virus. However, a recombinant rinderpest virus with insertion of eGFP at a similar location in the L protein grew like the wt virus in vitro but was attenuated for growth and virulence in its natural animal host, cattle (2
). Clearly, these data indicate that growth and virulence properties can be altered by insertion of eGFP into the viral polymerase. It will be interesting to examine whether VSV-PeGFP with insertion of eGFP in the P protein of the viral polymerase complex possesses altered growth and virulence phenotypes in vivo.
In cells infected with VSV-PeGFP, the intracellular distribution of fluorescence was punctate. The majority of these puncta were seen distributed throughout the cytoplasm, and their locations represented the sites of viral replication since the de novo-synthesized RNA as well as other components of viral replication machinery, namely, the N and the L proteins, colocalized to these sites. The interpretation that viral RNA synthesis occurs throughout the cytoplasm but in proximity to the cell nucleus was strengthened by the observation that in cells treated with NOC or Colcemid, which inhibit MT polymerization, leading to a disruption of nucleocapsid transport from the sites of synthesis to the cell periphery, the nucleocapsids were seen as aggregates around the nucleus (Fig. and ).
VSV contains approximately 450 copies of P molecules per nucleocapsid (51
). Although VSV-PeGFP contains almost half as many PeGFP molecules as there are P molecules in wt VSV (Fig. ), it still has more than the 120 copies of eGFP necessary to detect individual rotavirus particles (5
). Clearly, by live-cell imaging of infected cells at high magnification, we were able to detect individual nucleocapsids as tiny green fluorescent structures (Fig. and ). Many of these green structures were of uniform size, roughly approximating the size of viral nucleocapsids, although larger green dots, presumably representing multiple nucleocapsids in very close proximity, were also observed in drug-untreated cells. These green structures were not seen in cells expressing PeGFP alone or in combination with N and/or L proteins (data not shown). Many of these fluorescent nucleocapsids appeared to be associated with mitochondria and moved in parallel with the longitudinal axis of mitochondria toward the cell periphery. The significance of the association of the nucleocapsid with mitochondria is not clear at this time, but it is possible that cytoskeletal structures, like MTs, that are used by mitochondria for their intracellular distribution (18
) might be involved in nucleocapsid transport. The association of nucleocapsids with mitochondria may thus be transient and possibly serve as bridges linking MTs during transport of the nucleocapsids. Alternatively, it is possible that the association of nucleocapsids with mitochondria may be just random. Further work will be necessary to provide any functional significance to the association of nucleocapsids with mitochondria.
Although the effect of NOC on nucleocapsid distribution was dramatic (Fig. ), virus yield was reduced to about 20% of that from untreated control cells (Fig. ). It is possible that nucleocapsid transport may occur by additional mechanisms that are independent of MT. In this regard, it is of note that for DNA viruses whose capsids are specifically transported by MTs toward the cell periphery, a 20 to 25% reduction in virus yield in the presence of NOC has been considered to be significant (3
). In the light of these observations, our data are consistent with the interpretation that MTs are involved in the transport of viral nucleocapsids by an anterograde movement toward the cell periphery. Although the nucleocapsid transport toward the cell periphery was affected by NOC or Colcemid treatment, virus entry and uncoating remained unaffected, as seen by synthesis and accumulation of viral nucleocapsids with time in NOC-treated cells (Fig. ). This is consistent with the recent findings that NOC has no significant adverse effect on VSV entry, uncoating, or viral macromolecular synthesis (29
How might the MTs be involved in transport of viral nucleocapsids toward the cell periphery? Since the MTs form tracks on which cellular cargos are transported by intracellular kinesin and dynein motors (10
), it is possible to envision a scenario in which the nucleocapsids interact with kinesin and/or dynein motors and are transported by these motors on MT tracks. In this regard, it is interesting to note that the P protein of rabies virus (another rhabdovirus) has been shown to interact efficiently with the dynein light chain (14
), a component of the dynein motor. Although the significance of this interaction in terms of intracellular transport of the viral nucleocapsids has not been established, it is tempting to speculate that VSV nucleocapsids also interact with one or more components of the intracellular motors that play a role in the transport of the nucleocapsids mediated by MTs. With the availability of fluorescently tagged nucleocapsids, it will be possible to study these interactions and examine intracellular events by real-time imaging of virus-infected cells for a better understanding of the mechanisms by which the viral nucleocapsids are transported from the sites of synthesis to the assembly sites. Furthermore, VSV-PeGFP could be used to study entry and uncoating mechanisms that may provide significant insight into receptor-mediated endocytosis and uncoating of viral nucleocapsids in infected cells.