VSV recombinant PS > A4 was generated using reverse genetics [12,27] to contain four alanines in place of the PSAP motif within the M protein (Figure 1). For comparison, the corresponding amino acid sequence from VSV-wild type (VSV-WT), VSV host shutoff mutant (M51R), and VSV-PY > A4 (budding defective mutant containing four alanines in place of the PPPY motif within the M protein) are also depicted (Figure 1). M51R was used as a control in most experiments since the PS > A4 mutation was built into the M51R background (Figure 1). Our previous findings revealed that recombinant PS > A4 produced plaque size measurements and titers in BHK-21 cells that were virtually identical to those produced by both VSV-WT and M51R viruses [12,27]. In contrast, recombinant PY > A4 produced plaques with reduced diameters and titers that were on average 10-fold lower than those produced by control viruses [12,27].

We first sought to determine whether recombinant PS > A4 displayed an attenuated phenotype in a mouse model of VSV infection. Toward this end, groups of eight 6-week old BALB/c mice were infected with recombinant PS > A4 or with control viruses (VSV-WT or M51R) and then assessed for weight loss, survival, and virus replication in both the lungs and brain. Mice were inoculated with 10⁷ p.f.u. of the indicated virus and body weight was recorded on a daily basis for a period of 14 days (Figure 2). All mice infected with the parental M51R virus exhibited consistent and significant weight loss from day 0 to day 8, whereas mice that were mock-infected (PBS) or infected with either PS > A4 or PY > A4 lost <20% of their body weight and ultimately gained body weight during later days post‑infection (Figure 2).

All mice infected with the parental M51R virus eventually succumbed to the infection by day 9 post-infection (Figure 3). In contrast, all mice that were mock-infected or infected with VSV recombinants PS > A4 or PY > A4 survived during the 14 day period (Figure 3).

Three mice from each group were euthanized on day 2 for virus titration of lung and brain tissues using a standard plaque assay on BHK-21 cells (Figure 4). The mean viral titer of M51R in the lungs was 7.4 × 10⁶ p.f.u./mL whereas that of recombinant PS > A4 was approximately 7.0 × 10¹ p.f.u./mL (Figure 4). In brain samples taken from infected mice, M51R replication was detected in all three mice at an average titer of 3.2 × 10³ p.f.u./mL (Figure 4). Interestingly, replication of recombinant PS > A4 was undetectable in the brains from all three sacrificed mice (Figure 4). It should be noted that replication of recombinant PY > A4 was detected in the lungs of all three sacrificed mice at an average titer of 1.2 × 10⁵ p.f.u./mL (data not shown), and was detected in the brain of only 1 of 3 sacrificed animals (data not shown). The pathogenic nature of M51R may be related to the initial inoculum, age and strain of the mice, and/or a bolus of virus in the lung. These data indicate that mutagenesis of the PSAP motif to AAAA leads to a phenotype of severe attenuation compared to that of the parental virus in this small animal model of infection.

Since VSV is an arbovirus that must replicate in both an insect and mammalian host, we sought to determine whether recombinant PS > A4 also exhibited an attenuated phenotype in insect cells as compared to control viruses. Toward this end, mosquito C6/36 cells were infected with M51R, PY > A4, or PS > A4 at an MOI of 50, and virus released into the supernatant was collected at various times post-infection and quantified by standard plaque assay on BHK-21 cells performed in triplicate. To our surprise, we found that recombinant PS > A4 reproducibly replicated to titers in C6/36 cells that were up to 1.0 log greater than that of M51R, and up to 3.0 logs greater than that of recombinant PY > A4 (Figure 5). Titers for all three viruses steadily increased with time up to approximately 24 h.p.i., at which time titers of PS > A4 continued to rise to approximately 1.0 × 10⁸ p.f.u./mL at 70 h post-infection, whereas titers of M51R leveled off at approximately 1.0 × 10⁷ p.f.u./mL and titers of PY > A4 began to decline to approximately 1.0 × 10⁵ p.f.u./mL at 70 h post-infection (Figure 5).

In addition to using mosquito C6/36 cells, we also infected the KC cell line from Culicoides sonorensis, a known VSV vector species, with M51R and recombinant PS > A4 at an MOI of 3.0 (Figure 6). Consistent with the results obtained using C6/36 cells, recombinant PS > A4 replicated to titers approximately 10-fold higher in KC cells compared to those observed for M51R over a 72 h time period (Figure 6). Together, these data indicate that titers of recombinant PS > A4 were consistently higher than those of the parental M51R virus in insect cell lines.

Next, we used light microscopy to observe virus-induced CPE at 24 h.p.i. in monolayers of mosquito C6/36 cells infected with VSV-WT, M51R, or PS > A4 at MOIs of either 0.1, 1.0, 10, or 50 (Figure 7). There was no appreciable difference between CPE induced by VSV-WT or M51R viruses in C3/36 cells at all MOIs tested (Figure 7). In contrast, robust CPE was observed in cells infected with recombinant PS > A4 at the lower MOIs tested, and even more evident at MOIs of 10 and 50 (Figure 7). CPE induced by recombinant PS > A4 was dramatically distinct from that observed for VSV-WT or M51R viruses (Figure 7). Indeed, mosquito C6/36 cells infected with recombinant PS > A4 virus showed severe morphological alterations; including cell rounding, aggregation and detachment (Figure 7). Taken together, the titer and CPE data indicate that disruption of the PSAP motif of M results in a virus having enhanced growth properties and robust cytopathology in C6/36 and KC cells compared to those of parental control viruses: a phenotype completely opposite to that observed in a mammalian host.

To begin to address the mechanism by which recombinant PS > A4 establishes enhanced replication and cytopathology in insect cells, we sought to determine whether this cytopathic phenotype associated with recombinant PS > A4 may be linked to virus-induced apoptosis in mosquito C6/36 cells. While it is well established that VSV M plays a central role in the induction of apoptosis in mammalian cells in a caspase-3 dependent manner [4,5,6,20,21,36], a comparable role for M in insect cells is less well defined. Activated levels of caspase-3 were measured by colorimetric assay in mosquito C6/36 cells that were mock-infected, or infected with either VSV-WT, M51R, PY > A4, or PS > A4 at an MOI of 50 at 24 h p.i. (Figure 8). The level of activated caspase-3 induced by VSV-WT was normalized to 1.0 (Figure 8). As expected, the average level of activated caspase-3 induced by infection with M51R was virtually identical to that induced by VSV-WT (Figure 8). Similarly, the average level of activated caspase-3 induced by recombinant PY > A4 was within 2-fold of that observed for the parental M51R virus (Figure 8). In stark contrast to these results, the average level of activated caspase-3 induced by infection with recombinant PS > A4 was approximately 25-fold greater than that induced by VSV-WT and approximately 12.5-fold greater than that induced by M51R (Figure 8). These findings indicate that disruption of the PSAP motif dramatically affected the levels of activated caspsae-3 induced by VSV infection of mosquito C6/36 cells, and suggest that the enhanced cytopathology associated with recombinant PS > A4 may be linked to enhanced stimulation of the insect apoptotic pathway.

Aedes albopictus mosquito C6/36 cells were propagated in Leibovitz’s medium (Invitrogen; Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and penicllin/streptomycin (Invitrogen) and incubated at 28 °C under 5% CO₂. Culicoides sonorensis KC cells were propagated in Schneider’s Drosophila medium (Gibco, Grand Island, NY) containing 10% FBS at 30 °C. Wild-type (WT), M51R, PS > A4 and PY > A4 viruses have been described previously [12,13,24,28]. All VSV recombinants were propagated in BHK-21 cells and titrated by standard plaque assay on BHK-21 [27] or Vero cells [39].

Mosquito C6/36 cells in six-well plates were infected with the indicated viruses at MOIs of 0.1, 1.0 10 or 50. After incubation for 1 h at 28 °C, the medium was removed, and cells were washed with 1× phosphate-buffered saline (PBS) twice and incubated with fresh Leibovitz’s media containing 5% FBS at 28 °C. At 24 h p.i., cells were examined for CPE by light microscopy. For growth curves on C6/36 or KC cell lines, virions released into the supernatant at the indicated time points were quantified by titration using a standard plaque assay on BHK-21 cells [27].

Mosquito C3/36 cell in six-well plates were mock-infected (negative control) or infected with the indicated virus at an MOI of 50. After incubation for 1 h, the culture medium was removed, and cells were washed with 1 × PBS twice and then inoculated with fresh medium containing 5% FBS. At 24 h post-infection, approximately 5 × 10⁶ cells were harvested by centrifugation and washed two times with 1 × PBS. Cells were suspended in 50 μL of chilled Cell Lysis buffer (BioVision) and assayed using a Caspase-3 Colorimetric Assay kit (BioVision) according to the manufacturer’s instructions.

Groups of eight 6-week old female BALB/c mice were mock-infected with PBS, or infected intranasally with 10⁷ p.f.u./mL of VSV-WT, M51R, PY > A4, or PS > A4. Body weight measurements were recorded on a daily basis over a two-week period, and survival was monitored and plotted over a two-week period. At 2 days post-infection, three mice were sacrificed, and viral titers were determined from lung and brain homogenates of sacrificed animals using a standard plaque assay on BHK-21 cells.