Transport of cargo from the cell periphery towards the cell center often occurs along microtubules and is mediated by the minus-end-directed microtubule motor protein dynein. To analyze if transduction by rAAV2 is dependent on dynein function we overexpressed dynamitin in the cells to be infected. Dynamitin is a subunit of the dynactin complex and, if overexpressed, acts as a dominant negative inhibitor of dynein function by disrupting the coupling of cargo and the dynein complex. Thus, overexpression of dynamitin has been used to demonstrate dynein mediated intracellular transport of adenovirus and herpes simplex virus 1 (Dohner et al., 2006
; Suomalainen et al., 1999
). To allow us to measure dynamitin expression we generated an expression vector encoding for a fusion protein of dynamitin with the photostable red-fluorescent protein mCherry (Shaner et al., 2004
). A plasmid encoding for mCherry alone served as a negative control.
In a first step, we confirmed the expression of functional levels of dynamitin by visualizing the trans-Golgi network protein p230 (Gleeson et al., 1996
) in transfected cells 24 hrs post-transfection. As expected (Burkhardt, 1998
), non-transfected cells and cells expressing mCherry displayed a classical perinuclear localization of the Golgi, while the Golgi was dispersed throughout the cytoplasm in cells overexpressing dynamitin-mCherry ().
Overexpression of Dynamitin (p50) Has No Effect on rAAV2 Transduction
We then proceeded to determine the effect of dynamitin overexpression on AAV transduction. Twenty-four hours after transfection of HeLa cells with either a plasmid encoding dynamitin-mCherry or a plasmid encoding mCherry alone, we added 104 genome containing particles (gcp) per cell of rAAV2 encoding GFP. Twenty-four hours after transduction, mCherry and GFP expression were analyzed by FACS. As can be seen from , no difference in transduction could be detected between dynamitin-mCherry expressing and non-expressing cells. These results held true even for cells expressing the highest level of dynamitin-mCherry (data not shown). Similarly, overexpression of mCherry alone had no effect on transduction (). Furthermore, non-transfected control samples were transduced to an equal extent. These data indicate that, at least in HeLa cells, dynein-mediated transport is not mandatory for a productive transduction with recombinant AAV2. This does not exclude, however, that dynein-dependent transduction pathways exist in addition.
An alternative way to determine whether transport along microtubules is important in viral trafficking is the alteration of the microtubule polymerization state with drugs such as nocodazole, vinblastine and taxol.
Treatment of cells with nocodazole results in the disruption of microtubules (Jordan and Wilson, 1998
). Previous studies analyzing the effect of nocodazole on rAAV2 transduction yielded conflicting results. One study reported an inhibition of rAAV2 transduction by nocodazole (Sanlioglu et al., 2000
) while in a different study nocodazole had no effect (Alexander, Russell, and Miller, 1994
). In light of these discrepancies, we decided to study first the effect of nocodazole on rAAV2 transduction.
To this end, we pretreated HeLa cells for 4 hours with various concentrations of nocodazole followed by infection with 104
gcp/cell rAAV2-GFP. After incubation for 24 hrs in the continued presence of the drug, we analyzed GFP expression by FACS analysis. As reported by Russel and colleagues (Alexander, Russell, and Miller, 1994
), when using low concentrations of nocodazole, we observed no effect on AAV transduction. Conversely, as reported by others (Sanlioglu et al., 2000
), we observed an inhibition of transduction at higher concentrations of the drug when we looked at the total population of cells (data not shown).
However, we also noticed that high concentrations of nocodazole were very toxic () and that — as a result of the drastic change in morphology caused by the disruption of microtubules — it is very challenging to determine the viability of nocodazole treated cells based on the forward/side scatter (FSC/SSC) profiles alone (Fig. S1a
). Hence, we used an additional criterion for viability namely the exclusion of the live/dead cell dye 7-amino-actinomycin-D (7AAD). When we gated on cells that were alive — as determined by both their FSC/SSC profile and 7AAD exclusion (Fig. S1b
) — no inhibition of AAV transduction by nocodazole, even at the highest concentration tested, was observed (). Immunofluorescence microscopy revealed that in cells treated with nocodazole the microtubules were disrupted and the Golgi was, as expected (Burkhardt et al., 1997
), dispersed throughout the cytoplasm ().
Nocodazole Does Not Inhibit rAAV2 Transduction
Similar results were obtained with vinblastine, a drug that at low concentrations disrupts microtubules (Jordan and Wilson, 1998
) and is less toxic than nocodazole (). At concentrations of vinblastine that disrupt the microtubular network and disperse the Golgi (, left) no effect on rAAV2 transduction could be observed (). Interestingly, at higher concentrations of vinblastine, which result in the formation of tubulin paracrystals ((Manfredi and Horwitz, 1984
) and , right), vinblastine partially inhibits transduction ().
Vinblastine Has a Concentration-Dependent Effect on rAAV2 Transduction
We next wanted to determine if taxol, a drug known to stabilize microtubules (Jordan and Wilson, 1998
), has any effect on AAV transduction. For this, we pretreated HeLa cells for 4 hours with increasing concentrations of taxol and analyzed GFP expression 24 hrs after addition of 104
gcp/cell rAAV-GFP. shows that very high concentrations of taxol were, in addition to being less toxic than nocodazole ( vs. ), able to inhibit partially AAV2 transduction – even if dead cells were rigorously excluded. At these concentrations, tubulin formed randomly initiated bundles at the end of which Golgi fragments are located ( and (Wehland et al., 1983
High Concentrations of Taxol Partially Inhibit rAAV2 Transduction
It has recently been reported that AAV trafficking is dependent on the vector dose used (Ding et al., 2006
). To test if the role of microtubules in AAV2 transduction differs at various viral particle to cell ratios we analyzed the effect of microtubule-altering drugs at different vector doses. To allow us to use small viral particle to cell ratios in these experiments we took advantage of self-complementary AAV2 preparations encoding for EGFP. Because transgene expression with these vectors is not dependent on double-strand synthesis, they display an earlier onset of transgene expression and transduce cells more readily (Wang et al., 2003
). As can be seen from , the effect of microtubule-altering drugs is, to a certain degree, dependent on vector dose. At a particle to cell ratio of 100 the inhibition of transduction by 50 µM taxol (, solid bars) or vinblastine (, solid bars) were 60% and 69% respectively. On the other hand, at a gcp/cell ratio of 1,000 the relative reductions were approximately 45% and at a ratio of 10,000 only about 20% ( and , solid bars). At a concentration of 1 µM, taxol inhibited transduction as well and again more strongly at lower vector doses when compared to higher vector doses (, open bars). As for single-stranded AAV (10,000 gcp/cell), high concentrations of nocodazole (50 µM) had no effect on AAV2 transduction (, solid bars), regardless of the vector dose used. Low concentrations of nocodazole and vinblastine resulted in a slight inhibition of transduction (<20%) at the lowest vector dose ( and , open bars). The significance — if any — of this potential inhbition is currently unknown. Importantly, however, we never observed any inhibition at high concentrations of nocodazole supporting the conclusion that complete disruption of microtubules does not affect AAV2 transduction. We also observed that the inhibition of transduction by high concentrations of taxol and vinblastine at 104
gcp/cell was lower when we compare single-stranded and self-complementary virus (compare and ). The reason for this is likely that a smaller number of self-complementary vector particles is needed to result in a productive transduction because a potential rate-limiting step, i.e. double-strand synthesis, has been eliminated.
Effect of Microtubule-Altering Drugs on AAV2 Transduction at Various Vector Doses
Together, our data show that neither dynein function nor an intact microtubule network is necessary for a productive transduction of HeLa cells by rAAV2. On the other hand, the partial inhibition of transduction by high concentrations of taxol and vinblastine suggest that while intact microtubules are not required for transduction they might nevertheless play a complex role in rAAV2 transduction. Furthermore, the inhibitory effects of high concentrations of taxol and vinblastine are vector dose-dependent, the inhibition being most pronounced at low gcp/cell ratios. This behavior would be consistent with a model of multiple productive pathways for AAV2 transduction.