In the present study, we described a strategy for generating a dually fluorescent recombinant VSV. A VSV genome encoding PeGFP in place of wt P and Mtc in place of wt M allowed recovery of infectious virus. Cells infected with this virus and treated with the biarsenical red dye ReAsH produced progeny virions that appeared green under a fluorescence microscope due to incorporation of PeGFP and appeared red due to incorporation of ReAsH-labeled Mtc. This work describes for the first time the recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Using VSV encoding Mtc, we show by sequential labeling with the two biarsenical dyes that newly synthesized M protein reaches the plasma membrane within 30 min after synthesis. We further show that the transport and plasma membrane localization of M protein are not dependent on microtubules, which are generally used for transport of macromolecules inside the cell. Additionally, using the dually fluorescent virus, we show that following virus adsorption, it takes approximately 28 min for one-half of the virus particles to enter a cell and release the NCs into the cytoplasm in a synchronized infection model.
In the past few years, the use of fluorescent proteins as tags has been critical in understanding the dynamic events of virus infection in living cells (for reviews, see references 8
, and 23
). Using viruses genetically tagged with fluorescent proteins or with envelopes labeled with lipophilic dyes, several aspects of virus biology and virus-cell interactions have been examined. Fluorescent viruses have been used to study intracellular transport of viral capsids, capsid assembly, genome recombination, virus entry and egress, gene expression, tissue tropism and pathogenesis, and RNP transport and also for live tracking of single particles in infected cells (7
). For an enveloped virus such as VSV, which enters cells by endocytosis, infection proceeds by fusion of the viral envelope with the endosomal membrane, and subsequently, the NC is released into the cytoplasm. To study the early events during virus entry and the mechanisms of NC uncoating in the cytoplasm, dual-fluorescently labeled virus particles in which the NC core is labeled with one fluorescent color and the viral envelope is labeled with another fluorescent color would be highly desirable. Therefore, we sought to generate a dually fluorescent VSV where eGFP is fused in frame with the P protein (component of the viral NC core) and mRFP is fused in frame with the M protein (a component of the viral envelope). Multiple attempts to rescue such a virus failed, indicating that MmRFP protein with mRFP fused at the carboxy terminus of the M protein is not competent to support production of infectious VSV. Recently, similar attempts to recover rabies virus encoding a fluorescently tagged M protein were also unsuccessful (27
). Likewise, Ebola virus VP40 (the counterpart of matrix protein) fused with GFP also did not support virus-like particle assembly (45
). However, we were successful in generating a recombinant VSV encoding MmRFP as an extra cistron in the G-L intergenic junction (Fig. ). It should be noted that the fusion of mRFP to the M protein at either the carboxy terminus or the amino terminus retained the properties of M protein-like plasma membrane association and transcription inhibition of VSV minireplicons (S. C. Das, D. Panda, and A. K. Pattnaik, unpublished data), yet the M fusion proteins were not incorporated into the progeny virions. On the other hand, the Mtc protein with a small tag at the carboxy terminus of M faithfully recapitulated the properties of M protein and supported the assembly of infectious virus. It is possible that the large size of the MmRFP fusion protein interferes with the virus assembly functions of the M protein. Since the viruses encoding MmRFP in an extra cistron grew to reasonable titers (Fig. ), it appears that the MmRFP fusion protein does not impart a trans
-dominant-negative phenotype on the assembly functions of the wt M protein.
The method of tc tagging and biarsenical dye labeling of proteins in vivo was developed recently and holds promise to overcome the size constraints of fluorescent protein tagging (24
). One significant advantage of the system is that the tc hairpin can bind specifically and irreversibly to the biarsenical dyes immediately after synthesis, so the proteins can be visualized by fluorescence microscopy. The biarsenical labeling reagents can also be applied sequentially to label existing and nascently synthesized pools of proteins differentially and thus can allow for visualization of protein dynamics in living cells (20
). Therefore, tc tagging of M protein and sequential labeling of M with both dyes offer significant advantages over conventional detection of M protein with anti-M antibody staining. Using the two biarsenical dyes (ReAsH and FlAsH) and a sequential labeling approach, we have found that M reaches the plasma membrane within 30 min of synthesis and continues to accumulate there for 2 1/2 hours after synthesis (Fig. ). Previous biochemical and membrane fractionation studies have determined that plasma membrane association of the M protein is rapid and occurs less than 5 min after synthesis (2
). Although our data presented here are not as precise as those in the previous study (2
) due to the limitation of the biarsenical dye labeling method, which requires incubation with the dye for at least 30 min, our approach has been very useful in observing the protein dynamics of the existing and newly synthesized pools of M protein.
Previous studies with VSV (11
), rabies virus (41
), parainfluenza virus (13
), and measles virus (49
) have suggested that the matrix protein binds to NCs. Moreover, the M proteins of measles virus, parainfluenza virus, and Ebola virus drive budding of progeny virions by specific interaction with the viral NCs (13
). In cells infected with VSV-PeGFP-ΔM-Mtc and labeled with ReAsH, we were unable to observe interaction of M protein with the viral NCs either inside the cytoplasm or at the plasma membrane, consistent with previous reports (18
). It is possible that during virus assembly, initial interaction of a limited number of M molecules (beyond the limits of detection of fluorescence microscopy) with the NCs at the plasma membrane renders the NCs assembly competent, which then recruits more M protein to the assembly site. Thus, it appears that the mode of assembly of VSV might be different from that of other enveloped viruses at the plasma membrane.
The M protein of VSV is synthesized as a soluble fraction inside the cytoplasm and is transported toward the plasma membrane (2
). Early biochemical studies indicated that the M protein associates with the plasma membrane independent of VSV G protein (5
). However, the mode of transport of the M protein toward the plasma membrane is unclear. Since the M protein has been shown to physically associate with tubulin (42
), we reasoned that the M protein could be transported to the plasma membrane in a microtubule-dependent mechanism. However, our results (Fig. ) suggest a microtubule-independent mechanism for the transport of the M protein to the plasma membrane. Additionally, disruption of actin filaments, another cytoskeletal structure, also did not affect M protein transport (data not shown). Since we had previously shown that the viral NCs are transported toward the cell periphery in a microtubule-dependent manner (14
), our data presented here clearly support the idea that the M protein and the NCs are transported toward the plasma membrane independent of each other. Further work will be necessary to determine the mode of transport of the M protein to the plasma membrane.
During VSV infection, the M protein dissociates from other viral components and is distributed throughout the cytoplasm (47
). However, the step at which the M protein dissociates from the viral NCs is unclear. It has been demonstrated that fusion of the viral envelope with the endosomal membrane and the release of the NC into the cytoplasm are two independent but successive steps in the endocytic pathway of VSV infection (31
). Release of viral NCs into the lumen of the endosomal vesicle occurs by the fusion of the viral envelope with the membranes of the endosomes. However, the NC release into the cytoplasm may require a back-fusion event in which the internal vesicles fuse with the membranes of the late endosome (31
). These studies have shown that fusion of the endosomal membrane with the viral envelope occurs approximately 20 to 25 min after virus entry (31
). Using the dually fluorescent virus, our observation that NCs are released from virus particles with a half-life of uncoating of approximately 28 min suggests that the back-fusion event which is necessary for the release of the NCs into the cytoplasm following fusion of the viral envelope with the membranes of the endosomes must be a fast process requiring less than 10 min.
The potential use of the tc-tagged virus particles reported here for studies on virus entry and uncoating as well as virus assembly and egress in infected cells is exciting. The tc motif can be processed after ReAsH labeling for both live-cell imaging and photoconversion of diaminobenzidine to allow direct correlation of live-cell images with high-resolution electron microscopic data (20
). Thus, VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc viruses will be valuable tools in studies to understand the mechanisms of VSV assembly and the role of the M protein in the process. The precise location of the M protein inside the virion is unclear. Some studies suggest that the M protein forms a bridge between the G protein and the viral NC (11
), while other studies suggest that the M protein forms a cigar-shaped scaffold around which the viral NC is wrapped (3
). Since the ReAsH dye can be used for photoconversion of diaminobenzidine, which can allow direct correlation of live-cell images with electron microscopy (20
), the ReAsH-labeled VSV-ΔM-Mtc viruses could be used to reexamine the internal organization of the M protein in purified VSV.