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Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation (ΔL) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which we find are as nontoxic as second-generation ones: using longitudinal structural and functional two-photon imaging in mouse visual cortex in vivo, we found that they did not kill labeled neurons or perturb their response properties over the entire months-long courses of imaging. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in 25% - 525% increased numbers of retrogradely labeled neurons in vivo. These ΔL rabies viral vectors therefore constitute a new state of the art for nontoxic, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.


Introduction
Since their introduction to neuroscience in 2007 1,2 , recombinant rabies viral vectors have become widely-adopted tools in neuroscience, allowing "monosynaptic tracing" of direct inputs to genetically-targeted starting postsynaptic neuronal populations 2-4 as well as simple retrograde targeting of projection neurons when injected at the sites of these projection neurons' axonal arborizations 1,5 . These vectors are now used in a large number of laboratories worldwide and have contributed to many high-impact studies of a wide variety of neural systems [6][7][8][9][10][11][12][13][14] .
Because first generation ("∆G") rabies viral vectors (which have only the glycoprotein gene G deleted from their genomes) are cytotoxic 1,4,5,15 , we recently introduced second-generation, "∆GL" rabies viral vectors, which have both the glycoprotein gene G and the viral polymerase gene L (for "large" protein) deleted from their genomes 5 .
Because the viral polymerase is absolutely required for transcription of all genes from the rabies viral genome as well as for replication of the viral genome itself [16][17][18][19][20][21] , this additional deletion, by design, reduces gene expression to a minimal level (provided by the few starting copies of the polymerase protein that are copackaged in each viral particle) that appears to be completely harmless to the "infected" cells. Because transgene expression is reduced by the same degree, we inserted the genes for Cre and Flpo recombinase, of which even low levels of expression are sufficient to cause neuroscientifically-useful downstream effects such as expression of fluorophores or calcium indicators in labeled cells 5 . We originally showed that these ∆GL vectors are useful These findings that a ∆L rabies virus could be grown to 11-fold higher titer than a matched ∆GL one led us to predict that ∆L viruses would be superior tools for retrograde targeting in vivo, because their much higher titers would result in retrograde infection of many more projection neurons.

Retrograde targeting in vivo
To test this prediction, we made matched preparations of ∆GL and ∆L viruses expressing either Cre or Flpo (mouse-codon-optimized Flp recombinase 28 ), then injected each of the four viruses in the somatosensory thalami of reporter mice (Ai14 29 for the Cre viruses, Ai65F 30 for the Flpo one; both lines express tdTomato following recombination by the respective recombinase). We sacrificed the mice at either 7 days or 4 weeks after injection, sectioned and imaged the brains by confocal microscopy, and counted the numbers of retrogradely labeled cells in cortex. Fig. 2 shows the results.
As seen in panels 2b-e, for both recombinases, and at both timepoints, the ∆L viruses significantly outperformed the ∆GL ones.
For the Flpo viruses, the difference was dramatic: at the 1-week timepoint, the ∆L virus labeled 24 times as many cells as the ∆GL one (although this difference was not statistically significant due to high variance in the ∆L cohort: single factor ANOVA, p=0.275, n = 8 mice each group); by the 4-week timepoint, the ∆L-Flpo virus had labeled 6.25 times as many cells as the ∆GL counterpart, a difference that was extremely significant (single factor ANOVA, p=3.21E-04, n = 8 mice each group). For the Cre viruses, the difference was smaller, presumably due a ceiling effect (see Discussion), but still highly significant: at 1 week, the ∆L-Cre virus had labeled 1.40 times as many cells as ∆GL-Cre (single factor ANOVA, p=4.20E-03; n = 4 mice each group); at the 4-week timepoint, the ∆L-Cre virus had labeled 1.25 times as many cells as ∆GL-Cre (single factor ANOVA, p=7.38E-04, n = 4 mice per group).
We also made some injections of RV∆L-Cre (in thalami of Ai14 mice) with the much longer survival times of 4 or 6 months (Fig 2f-g). The results at both of these longer survival times appeared very similar to those at the shorter ones. Consistent with extensive prior literature on corticothalamic neurons 31-33 and with our previous results with corticothalamic injections of ∆G and ∆GL viruses 1,5 , the cells labeled in cortex by both viruses at all timepoints were pyramidal neurons in layer 6, with a few in layer 5. Furthermore, labeled neurons all appeared morphologically normal even months after injection, with the fine processes of axons and dendrites, including individual spines (rightmost images in 2f-g) clearly visible and without blebbing or other obvious abnormalities.
As a further test of the flexibility of ∆L vectors, we made a version expression the tetracycline transactivator (tTA) and injected it in the thalamus of Ai63 reporter mice (in which TRE-tight drives tdTomato expression) 30 . As seen in Extended Data Fig. 2, thousands of cortical thalamic cells were found retrogradely labeled at both 1-week and 4-week survival times, with no significant difference between the numbers at the two timepoints (single factor ANOVA, p = 0.772, n = 4 mice per group).
(see Supplementary File 3 for all counts and statistical comparisons. See also Supplementary Video 1 for a rendering of a group of ∆L-labeled neurons at 2 weeks and again at 10 weeks.

Longitudinal functional two-photon imaging in vivo
We went on to examine the functional properties of RV∆L-labeled neurons in vivo. As for the structural imaging (see above), we injected RV∆L-Cre in the primary visual cortices of reporter mice, in this case mice that express the calcium indicator GCaMP6s 34 after Cre recombination (Fig. 4). Beginning one week later, we began imaged the calcium signals in the labeled neurons in a series of imaging sessions that continued until 16 weeks postinjection, in the awake mice viewed visual stimuli consisting of drifting gratings of different orientations and frequencies. Just as we found previously for ∆GL viruses 4,5 , cells labeled by the third-generation vector showed no signs of dysfunction for as long as we followed them (

Discussion
Here we have shown that deletion of just the polymerase gene renders rabies viral vectors nontoxic, like secondgeneration (∆GL) vectors, but makes them much more efficient at replicating within complementing cells in culture. This ability to be grown to much higher titers results in significantly increased transduction of projection neurons within a given pathway. This more comprehensive access to projection neurons will increase the yield and efficacy of systems neuroscience experiments that depend on the retrograde targeting approach.
In the corticothalamic pathway that we have examined here, the advantage of a ∆L vector over the ∆GL equivalent was clearest in the case of the Flpo-expressing versions, with the ∆L vector labeling 6.25 times as many neurons as the ∆GL one did at four weeks postinjection. This ratio is of the same order of magnitude as the ratio of the titers of the injected Flpo viruses (14.4: see Methods). By contrast, for the Cre-expressing versions, the advantage of the ∆L vector over the ∆GL one was more modest, labeling 1.25 times as many cells, even though the ratio of the titers of these Cre vectors was even higher (20.5). Because the absolute numbers of retrogradely labeled neurons, as well as the titers, were much higher for the Cre viruses than for the corresponding Flpo ones, we presume that the smaller advantage of the ∆L version seen in this case was because of a ceiling effect, with the ∆GL-Cre virus already labeling most of the available neurons in this pathway.
One could certainly argue that the much higher titers that we are easily able to obtain with ∆L vectors could also, in theory, potentially be achieved with ∆GL vectors, if enough effort were put into generating and testing producer cell lines expressing both G and L in order to find one that expressed the two genes at just the right ratio and levels. In practice, however, this hypothetical future research effort does not detract from the fact that the best currently-existing preparations of ∆L rabies viral vectors have much higher titers than the best currently-existing preparations of ∆GL ones, making them better tools for retrograde targeting.
We note that, although here we have only demonstrated the use of ∆L rabies viral vectors in mice, they are also highly likely to work in a wide variety of mammalian species, because, apart from their shorter RNA genomes, the structural properties of third-generation (and second-generation) rabies viral particles are identical to those of first-generation ones, which have been successfully used in diverse mammalian species including rats 35 , cats 36,37 , ferrets 38 , and macaques 12,39-45 (and even in fish 46-48 and frogs 49 ).
Our findings here that ∆L rabies viruses have extremely low expression levels and do not replicate within (or spread beyond, in vivo) non-complementing cells are entirely consistent with similar findings in cell culture in a recent report on an L-deficient rabies virus encoding firefly luciferase 50 .
A note about safety: our results strongly suggest that ∆L rabies viruses are completely unable to replicate in the absence of complementation and moreover are harmless to any cells that they transduce. However, a mixture of ∆L and ∆G viruses could pose a safety risk, because such viruses will be mutually complementary. Care must therefore be taken to avoid contamination between ∆L and ∆G constructs -either packaged viruses or the genome plasmids used to make them -which would have the potential to create a self-complementing replication-competent mixture (see Hidaka et al. 51 for an example of such a self-complementing mixture). Finally, we have recently shown 4 that second-generation (∆GL) rabies viral vectors can spread transsynaptically when complemented by provision of both G and L in trans. That is, complementation of an Ldeficient rabies virus (even a G-and L-deficient virus that is complemented by G) allows it to spread beyond initially infected cells in vivo. It is therefore reasonable to infer that provision of L in trans should allow thirdgeneration, ∆L rabies viral vectors to spread beyond initially infected cells, given that we have shown here that such complementation in cell culture allows ∆L viruses to replicate very efficiently. We have also shown here, with the longitudinal two-photon imaging of labeled neurons, that ∆L viruses do not spread beyond initially infected cells in vivo in the absence of complementation. Collectively, our results therefore suggest the outlines of a third-generation monosynaptic tracing system based on ∆L vectors complemented with L expression in trans. However, genetic targeting of a ∆L vector to specific starting cell types might appear elusive: in the firstand second-generation systems 2,4 , this targeting is achieved by packaging the rabies viral particles with an avian retroviral envelope protein (EnvA) instead of its own envelope glycoprotein, so that they can only infect cells that have been engineered to express EnvA's cognate receptor. On the face of it, this pseudotyping strategy requires that G be deleted from the rabies viral genome, because expression of G by the virus within the EnvA-expressing producer cells would result in the production of virions with membranes populated by a mixture of EnvA and the rabies viral glycoprotein. If this challenge could be overcome by sufficiently clever means, our present findings that ∆L viruses replicate more readily in complementing cells, which is the fundamental process central to monosynaptic tracing 2 , suggest that a third-generation monosynaptic tracing system could be much more efficient than the second-generation one.

Methods
All experiments involving animals were conducted according to NIH guidelines and approved by the MIT Committee for Animal Care. Mice were housed 1-5 per cage under a normal light/dark cycle for all experiments.

Cell lines
The BHK-B19G3 cell line, expressing the SAD B19 strain rabies virus glycoprotein gene, was made by resorting BHK-B19G2 cells 55 on a BD Facs Aria cell sorter and retaining the brightest 2% of EGFP-positive cells as well as the next-brightest 18%. Following the sort, both populations were expanded and refrozen, then thawed and tested for their efficacy at supporting replication of ∆G virus; the second-brightest population ("BHK-B19G3_2") was found to result in higher titers and is referred to here as BHK-B19G3.
The BHK-B19L cell line, expressing the SAD B19 strain rabies virus polymerase gene, was made by transfecting BHK-21 cells (ATCC CCL-10) with pCAG-hypBase 15 and pB-CAG-B19L-IRES-mCherry-WPRE-BGHpA 4 using Lipofectamine 2000 (Thermo Fisher 11668019), then expanding the cells and sorting on a FACS Aria sorter (BD) to collect the brightest 5%, as well as the next brightest 5%, of mCherry-expressing cells. The two collected populations were expanded and refrozen, then thawed and tested for their efficacy at supporting replication of ∆L virus; the second-brightest population ("BHK-B19L_2") was found to result in higher titers and is referred to here as BHK-B19L.
The BHK-B19L-G cell line, expressing the SAD B19 strain rabies virus polymerase and glycoprotein genes, was made by transfecting BHK-B19L cells (see above) with pCAG-hypBase and pB-CAG-B19G-IRES-EGFP-WPRE-BGHpA (see above), then expanding and sorting on a BD FACS Aria, keeping the brightest 5%, as well as the next brightest 5%, of EGFP-expressing cells which also expressed mCherry. The sorted cells were expanded and refrozen, then thawed and tested for their efficacy at supporting replication of ∆GL virus; the brightest population ("BHK-B19L-G_1") was found to result in higher titers and is referred to here as BHK-B19L-G.
The 293T-TREtight-EGFP cell line for titering tTA-expressing viruses was made by transfecting HEK 293T/17 cells with pCAG-hypBase and pB-TREtight-EGFP (described above), then expanded and sorted on a BD FACS Aria, excluding the brightest 2% of EGFP cells, and keeping four of the next brightest EGFP cell populations. The sorted cells were expanded, frozen, and then thawed for testing their efficacy at titering ∆L-tTA virus. The fourth-brightest tranche of cells was used for subsequent titering of ∆L-tTA virus.

Rabies virus production and titering
The first-generation vector RV∆G-4Cre, the second-generation vectors RV∆GL-4Cre and RV∆GL-4Flpo, and the third-generation vectors RV∆L-5Cre, RV∆L-5Flpo, and RV∆L-5tTA were rescued as described previously 5 using genome plasmids pRV∆GL-4Cre, pRV∆GL-4Flpo, pRV∆L-5Cre, pRV∆L-5Flpo, and pRV∆L-5tTA, respectively. For simplicity, these viruses are referred to in this manuscript as RV∆G-Cre, RV∆GL-Cre, RV∆GL-Flpo, RV∆L-Cre, RV∆L-Flpo, and RV∆L-tTA, omitting the numbers denoting the positions of the transgenes within the viral genomes. Rescue supernatants were collected and filtered as described 56 , titered on the reporter cell lines 293T-FLEX BC (for Cre viruses) or 293T-F14F15S-BC (for Flpo viruses) 15 as described 55 , then used to infect BHK-B19G3, BHK-B19L-G, or BHK-B19L cells (see above) at multiplicities of infection ranging from 0.1 to 1. Supernatants from these "P1" plates were collected and titered as described 56 ; in some cases, these were used for a similar second passage ("P2"). Purification and concentration of either P1 or P2 supernatants was as described 55 , with supernatants treated with benzonase (Sigma 71206) (25 minute incubation at 37°C with 30 units/ml at) before ultracentrifugation. Concentrated viruses were aliquoted and frozen at -80°C. Rabies viruses were titered on reporter cells (293T-FLEX-BC for Cre viruses, 293T-F14F15S-BC for Flpo viruses, 293T-TREtight-EGFP (see above) for RV∆L-tTA) as described 55 , using a LUNA-II cell counter (Logos Biosystems) instead of a hemocytometer for counting cells, and in some cases using two-fold (as opposed to ten-fold) dilution series for more precise comparisons of titers.
Immunostained cells on coverslips were mounted on slides using Prolong Diamond Antifade mounting medium (Thermo P36970) and imaged on a Zeiss LSM 900 confocal microscope using a 20x objective.
For matched flow cytometric analysis of immunostained cells, cells were plated in 24-well plates without poly-Llysine-coated coverslips but otherwise immunostained as described above, then analyzed on an LSR II flow cytometer (BD) using FACS Diva software (BD). Histograms displayed in Fig. 1 were smoothed using the FACS Diva "Smooth histogram" setting; histograms in Extended Data Fig. 5 were not smoothed.

Viral growth analysis
For determining growth curves, BHK-B19G3, BHK-B19L-G, and BHK-B19L cells (see above) were plated in 10 cm plates coated in poly-L-lysine in normal medium (10% fetal bovine serum (VWR 16777-014) and antibiotic-antimycotic (Thermo 15240096) in DMEM (Thermo 11995073)) 55 . The following day, cells were infected with RV∆G-4Cre(B19G), RV∆GL-4Cre(B19G), or RV∆L-5Cre(B19G) at an MOI of either 1 (for singlestep growth curves) or 0.01 (for multi-step growth curves), with viruses diluted in normal medium at a total volume of 2 ml per plate, with each condition in triplicate. Following a one-hour incubation, the virus-containing medium was aspirated, plates were washed twice in DPBS (Thermo 14190144), and 12 ml fresh medium was added to each plate before they were returned to the incubator. Every 24 hours for the following five days, 200 µl of supernatant was collected from each plate; these supernatant samples were filter-sterilized using a 96well 0.45um PVDF filter plate (Millipore MSHVN4510), then frozen at -80°C before all samples were thawed and titered on HEK 293T-FLEX-BC cells as described above.

Mouse strains
The Cre-dependent tdTomato reporter line Ai14 29 was purchased from Jackson Laboratory (catalog # 007914). For mice to be used for two-photon imaging, a 3 mm craniotomy was opened over primary visual cortex (V1). Glass windows composed of a 3mm-diameter glass coverslip (Warner Instruments CS-3R) glued (Optical Adhesive 61, Norland Products) to a 5mm-diameter glass coverslip (Warner Instruments CS-5R) were affixed over the craniotomy with Metabond (Parkell) after virus injection.
Perfusions, histology, and confocal imaging 1 week to 6 months (see main text) after injection of rabies virus, anesthetized mice were transcardially perfused with 4% paraformaldehyde. Brains were postfixed overnight in 4% paraformaldehyde in PBS on a shaker at 4°C and cut into 50 μm coronal sections on a vibrating microtome (Leica, VT-1000S). Sections were collected sequentially into 6 tubes containing cryoprotectant, so that each tube contained every sixth section, then frozen at -20°C. Sections to be imaged were washed to remove cryoprotectant, then mounted with Prolong Diamond Antifade mounting medium (Thermo Fisher P36970) and imaged on a confocal microscope (Zeiss, LSM 900). To ensure that the confocal images included in the figures are representative of each group, the images were taken after the counts were conducted, and the mouse with the next higher number of labeled neurons than the average number for its group was selected for confocal imaging.

Quantification of retrograde targeting
Coronal sections between 0.43mm anterior and 4.07mm posterior to bregma were imaged with an epifluorescence microscope for cell counting (Zeiss, Imager.Z2). Due to the high density of retrogradely labeled tdTomato neurons in the cortex at the injection site (VPM/Po), cells were counted using the Analyze Particle function in ImageJ (size in micron^2: 20-400; circularity: 0.20-1.00). Only one of the six series of sections (i.e., every sixth section: see above) was counted for each mouse. P-values for all comparisons were obtained using single-factor ANOVAs.

Structural two-photon imaging and image analysis
Beginning seven days after injection of each rabies virus and recurring at the subsequent indicated timepoints (see main text) up to a maximum of 16 weeks following rabies virus injection, fields of view (FOVs) were imaged on a Prairie/Bruker Ultima IV In Vivo two-photon microscope driven by a Spectra Physics Mai-Tai Deep See laser with a mode locked Ti:sapphire laser emitting at a wavelength of 1020 nm for excitation of tdTomato.
In order to distinguish individual labeled neurons, FOVs were chosen some distance away from the area of brightest tdTomato labeling. Two well-separated areas were chosen in each mouse. For each imaging session, mice were reanesthetized and mounted via their headplates to a custom frame, with ointment applied to protect their eyes and with a handwarmer maintaining body temperature. Imaging parameters were as follows: image size 512 X 512 pixels (282.6 μm x 282.6 μm), 0.782 Hz frame rate, dwell time 4.0 μs, 2x optical zoom, Z-stack step size 1 μm. Image acquisition was controlled with Prairie View 5.4 software. Laser power exiting the 20x water-immersion objective (Zeiss, W plan-apochromat, NA 1.0) varied between 20 and 65 mW depending on focal plane depth (Pockels cell value was automatically increased from 450 at the top section of each stack to 750 at the bottom section). For the example images of labeled cells, maximum intensity projections (stacks of 100-200 μm) were made with ImageJ software. Cell counting was automated using the "Analyze Particles" function in ImageJ. Plots of cell counts were made with Prism 9 (GraphPad Software, San Diego, California).

Functional two-photon imaging and image analysis
For functional two-photon imaging of RV∆L-Cre-labeled cells, FOVs were slightly offset from the regions of brightest GCaMP6s label in left-hemisphere V1 in order to allow separate identification of individual cells. This imaging was performed using the same microscope (5.356-Hz frame rate, 1024X 128 pixels, 565.1 μm x 565.1 μm, dwell time 0.8 μs, 1x optical zoom, scan angle 45 degree) with the same objective and laser (at 920 nm) as for the structural imaging experiments. Laser power at the objective ranged from 10 to 65 mW. Calcium imaging data were acquired in supragranular layers (100 to 200 μm deep). Surface vasculature provided coarse fiducial markers for finding the same FOVs in different imaging sessions.
For these experiments, mice were awake and head-fixed. No behavioral training or reward was given. Visual stimuli were generated in Matlab (R2015R version) with custom software based on Psychtoolbox (http://psychtoolbox.org) and shown on the same LCD screen as in the widefield mapping experiments. Each condition consisted of 2 s of a full-field sine wave grating drifting in one direction, presented at 80% contrast with spatial frequency of 0.04 cycles/ degree, followed by 2 s of uniform mean luminance (gray). All permutations of 12 directions (30° steps) and 5 temporal frequencies (1, 2, 4, 8 and 15 Hz) were shown, in randomized order. The complete set was repeated 10 times, for a total stimulation period of 40 min per FOV per session. Cells were then manually segmented, and single-cell fluorescence traces were extracted by averaging the fluorescence of all pixels masking the soma, using ImageJ (version 2-0-0-rc-69) software. . The mean ΔF/F over the full 2 s of each stimulus condition was used to calculate orientation tuning curves, with background fluorescence (F) in Δ F/F taken as the value of the trace immediately preceding a condition, averaged over all conditions. The raw calcium traces from cells within individual FOVs (not across FOVs, given different imaging conditions across animals and time points) were sorted by mean fluorescence. Randomly colored ROI view images were created by suite2p (https://www.suite2p.org). For 'tuned' cells in Figure 4 panel f and g, the counts are based on all imaged neurons' individual tuning curves, plotted in MATLAB; any cell showing response to a preferred orientation (including narrowly tuned neurons and broadly tuned neurons) at any temporal frequency (1Hz, 2Hz, 4Hz, 8Hz, or 15Hz) was counted manually as a tuned cell.

Data availability
All cell counts and statistical analyses are provided in Supplementary Information. The novel plasmids described in this paper have been deposited with Addgene with the accession numbers given in Methods.  Fig. 1: Rabies virus with just the polymerase gene deleted (∆L) is phenotypically similar to doubledeletion-mutant (∆GL) virus but replicates to much higher titers within complementing cells. a-d, Deletion of just the polymerase gene L reduces transgene expression to levels that are very low but still sufficient to support reporter allele recombination in Cre reporter cells. a, Negative controls (uninfected cells). Top: Uninfected HEK 293T cells stained for rabies virus nucleoprotein (green) and for Cre (red). Histograms to right of panels show flow cytometric quantification of baseline fluorescence of uninfected cells in these channels. Bottom: Uninfected reporter cells which express mCherry following Cre recombination. Little signal is seen in these negative controls. b, Cells infected with a first-generation (∆G) vector expressing Cre. Both Cre and N are expressed at very high levels, and infected Cre reporter cells brightly express mCherry (note that dilutions at which roughly half of cells were infected were chosen for this figure). c, Consistent with our previous findings 5 , expression of both nucleoprotein and Cre from a second-generation (∆GL) vector is drastically reduced with respect to the first-generation vector, with expression levels comparable to those seen in negative controls. Despite this, the low Cre levels are still high enough to activate mCherry expression in reporter cells. d, A third-generation (∆L) vector expresses nucleoprotein and Cre at similarly very low levels, but again Cre expression is nonetheless high enough to successfully activate mCherry expression in reporter cells. e-f, Third-generation (∆L) vectors grow to much high titers in cultured cells than second-generation (∆GL) ones do. e, Viral titers in supernatants of complementing cells (expressing L, G, or both) infected with ∆L, ∆GL, or ∆G viruses at a multiplicity of infection (MOI) of 0.01 ("multi-step growth curves"), with supernatants collected every 24 hours for five days. Whereas a ∆GL virus only achieves 1.03E+06 infectious units (i.u.)/ml over the duration of the experiment, the ∆L virus grows to 6.25-fold higher on the same cell line, and 12.5-fold higher on a line expressing L alone. The highest ∆L titers obtained in this experiment were significantly higher than the highest obtained with a first-generation (∆G) virus (single-factor ANOVA, p = 2.30E-03, n = 3 replicates per condition). f, Similarly, at a MOI of 1 ("single-step" growth curves), the ∆GL virus titer peaks at 2.34E+06 i.u./ml, whereas the peak titer of the ∆L virus is 2.58E+07 i.u./ml, 11.0-fold higher than that of the ∆GL virus and not significantly different from that of the ∆G virus (single-factor ANOVA, p = 0.113, n = 3 replicates per condition). Graphs in e f show means ± s.e.m.   g, Comparison of the percentages of labeled cells that were visually tuned at 2 weeks and 14 weeks. The percentages increased moderately but significantly between the two timepoints, from 60% to 68% (paired twosample t-test, p = 0.0178, n = 6).