Cultures infected with a single baculovirus
Insect cell cultures individually infected with bacGFPVP2 or VP6 were analyzed by flow cytometry at 24 and 48 hours post-infection (hpi). MOI of 0.1, 1, 5, 10, and 20 pfu/cell were tested. Fluorescence histograms at 24 hpi are shown in Figure 1, along with an uninfected control culture performed simultaneously. When analyzing these results, it should be taken into account that fluorescence emission was measured at two different wavelengths, 510 for GFPVP2 and 575 nm for immunolabeled gp64 and VP6. Thus, background fluorescence of uninfected cells was different. Cells expressing a recombinant protein could be easily distinguished from uninfected cells. The distribution of the populations expressing GFPVP2 was different from the one expressing VP6. Data extracted from fluorescence histograms of duplicate cultures are summarized in Figure 2A, where the fraction of the population expressing a recombinant protein is reported relative to the total cell count (regardless of viability). It should be considered that the analysis shown in Figure 1 allows the identification of the population expressing a recombinant protein (either GFPVP2 or VP6), but not necessarily that of infected cells, as expression of the recombinant gene might be absent or inefficient even in infected cells. A marker that has been used to monitor infection by baculovirus is gp64 [22]. gp64 is the major envelope glycoprotein of baculovirus [23], and accumulates in the membrane of infected cells during the first 10 to 12 hpi [24]. To correlate infection with recombinant protein expression, gp64 was immunodetected at 24 hpi in cultures expressing GFPVP2 (Figures 1 and 2A). In addition, the population distribution predicted by Poisson:
commonly used to describe infection, is also plotted in Figure 2A[3]. The population fraction expressing any of the recombinant proteins or gp64 increased in a hyperbolic-type function with MOI up to 5 pfu/cell, and then remained constant for MOI above 5 pfu/cell. Such a behavior is in agreement with predictions based on the Poisson distribution. Cultures infected with bacVP6 or bacGFPVP2 had a similar trend, which was also similar to the population expressing gp64 in the cultures infected with bacGFPVP2. The similar behavior between cells expressing GFPVP2, gp64 and VP6 confirms that the results obtained from cells immunostained for VP6 were representative of the population expressing that recombinant protein, and that both recombinant baculoviruses were equally infective. Results obtained at 48 hpi are shown in Figure 2B. The Poisson distribution is not plotted in Figure 2B, as secondary infection had occurred at this time, and different calculations are needed to predict the infected population under these conditions (see discussion below). At 48hpi, between 65 to 90% of the cells were expressing either recombinant protein, regardless of the MOI. However, at MOI below 10 pfu/cell, a smaller population of cells expressed GFPVP2, in comparison with that expressing VP6.
Handling of cultures expressing GFPVP2 for FACS analysis was easier than those for VP6, as VP6 had to be immunostained by a laborious procedure, whereas intrinsic fluorescence of GFPVP2 facilitated the assay. Therefore the population containing this recombinant protein was followed every 24 h until 96 hpi. Results are shown in Figure 3A. The population expressing GFPVP2 reached a maximum at 24 hpi for cultures infected at a MOI of 5 pfu/cell or higher. In contrast, such population reached a maximum until 48 hpi for cultures infected at MOI of less than 5 pfu/cell. In all cases, the percentage of the population expressing a recombinant protein decreased after 48 hpi (Figure 3B), probably due to degradation of the recombinant protein after loss of cell membrane integrity in non-viable cells. Viability decreased steadily after infection, reaching 0% at 72 hpi in cultures infected at MOI of 10 and 20 pfu/cell. The rate of decrease of the viability was lower as the MOI decreased.
Cultures simultaneously infected with bacGFPVP2 and bacVP6
Gating parameters used for discriminating between cells expressing GFPVP2, VP6 or both proteins were set according to fluorescence (emission) at 510 nm (corresponding to GFP) and 575 nm (corresponding to R-phycoerythrin, used to detect VP6) in uninfected cultures, cultures expressing only GFPVP2, or cultures expressing VP6. Typical results are shown in Figures 4A–C. As expected, uninfected cultures had a low emission both at 510 or 575 nm (Figure 4A), whereas cells infected with bacGFPVP2 had a high emission at 510 nm (Figure 4B). Moreover, cells infected with bacVP6 had a high fluorescence at 575 nm (Figure 4C). Accordingly, gating parameters, which allowed the differentiation between the three populations present in coinfected cultures, could be set. Flow cytometry results of a culture simultaneously infected with bacGFPVP2 and VP6 at 24 or 48 hpi are shown in Figure 4D and 4E, respectively. At 24 hpi many cells could be clearly classified as expressing VP6, whereas only relatively few cells were expressing both recombinant proteins. At 48 hpi, only a few cells were not expressing a recombinant protein, while most were either expressing VP6 or both recombinant proteins simultaneously. A small fraction of the population was expressing only GFPVP2. A quantitative analysis of the flow cytometry study is presented below.
The results obtained from 4 experimental setups performed in duplicate are summarized in Figure 5. Cultures were infected at different MOI combinations of bacGFPVP2 and bacVP6. MOI of each virus were not increased above 5 pfu/cell, as higher MOI did not result in a higher population fraction expressing any of the recombinant proteins in single infections (Figure 2). In most cases, the largest fraction of the population expressed VP6, regardless of the MOI of bacGFPVP2. The only exceptions were the cultures infected with 0.1 and 5 pfu/cell of bacVP6 and bacGFPVP2, respectively, either at 24 or 48 hpi. In these cultures, the population expressing either of the recombinant proteins was about the same. It should be noted that, in the extreme situation, the population fraction expressing VP6 was 17.5 times larger than that expressing GFPVP2 (MOI bacVP6 1 pfu/cell, MOI bacGFPVP2 0.1 pfu/cell, 24 hpi). The highest percentage of the population expressing any recombinant protein (VP6 or GFPVP2 or both) at 24 hpi was obtained at MOI of 1 pfu/cell for both bacGFPVP2 and bacVP6, i.e. a total MOI of 2 pfu/cell. Such population fraction was 15% higher than that expressing any recombinant protein at the highest total MOI tested, which corresponded to 6 pfu/cell (5 pfu/cell of bacGFPVP2 and 1 pfu/cell of VP6). The population fraction expressing both recombinant proteins at 24 hpi closely followed the population expressing GFPVP2.
Coinfections had various effects on the population expressing GFPVP2. In general, the cell population expressing GFPVP2 in coinfected cultures was smaller than in single-infected cultures at the same MOI, except in two cases at 24 hpi (Figures 2 and 5). Namely, in cultures infected at a MOI of 0.1 pfu/cell of each baculovirus, the population expressing GFPVP2 was duplicated, in comparison to that observed in single infections. Moreover, at 0.1 pfu/cell of bacVP6 and 1 pfu/cell of bacGFPVP2, the population expressing GFPVP2 was the same than that observed in single infections. In contrast, the population expressing VP6 at 24 hpi increased in coinfections, compared to individual infections. In cultures infected at MOI of 0.1 pfu/cell of bacVP6, 11.5% of the population expressed VP6 in single-infected cultures, whereas in coinfections up to 45% of the population expressed VP6 (Figures 2 and 5). Something similar occurred at a MOI of bacVP6 of 1pfu/cell, as 65% of the population expressed VP6 in single-infected cultures, while in coinfections such percentage increased to 70 – 85% at 24 hpi.
Compared to 24 hpi, the population expressing any of the recombinant protein increased at 48 hpi (Figure 5). This increase occurred even in cultures infected with a MOI of bacGFPVP2 of 5 pfu/cell; a behavior not observed in individual infections. The population fraction expressing VP6 at 48 hpi reached 98% (considering the error bars) in cultures infected at MOI of 0.1 or 1 pfu/cell of any of the baculoviruses. In most cases the percentage of the population expressing GFPVP2 at 48 hpi was smaller than that expressing VP6, with the exception of the cultures infected with 0.1 pfu/cell of bacVP6 and 5 pfu/cell of bacGFPVP2, where the population expressing either protein was equal. The condition that resulted in the highest percentage of the population simultaneously expressing both recombinant proteins was a MOI of bacGFPVP2 of 5 pfu/cell and 1 pfu/cell of bacVP6. Such condition resulted in 48% and 58% of the population at 24 and 48 hpi, respectively, simultaneously expressing both recombinant proteins. Populations expressing the recombinant proteins were not followed after 48 hpi as further infections by progeny viruses were not expected after 48 hpi, and a rapid decline in viability was observed after this time (data not shown).
To better appreciate the effect of coinfections, the population expressing any of the recombinant proteins (GFPVP2, VP6, or both) at 24 hpi is shown in Figure 6 as a function of total MOI (MOI bacGFPVP2 + MOI bacVP6). For comparison, the Poisson distribution is also plotted. It can be seen that coinfected cultures with a MOI of bacVP6 equal or higher than that of bacGFPVP2 followed the Poisson distribution. However, cultures where the MOI of bacGFPVP2 was 5, 10, or 50 times higher than that of bacVP6 had a much lower population expressing any of the recombinant proteins than that predicted by the Poisson distribution at the corresponding cumulative MOI.