Sunday, June 4, 2023

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis – Nature Communications

RNA-seq analyses of adult and weanling brain microvessels identify candidate gene regulators of LACV infection

We first sought to identify candidate gene regulators that contribute to age-dependent susceptibility to LACV. Weanling (W) and adult (A) mice were treated with either poly I:C (PI) for 3 h (as a surrogate for a viral RNA stimulus) or vehicle control (V). Whole brain microvessel fragments isolated from these mice were subjected to RNA-seq analysis to identify both intrinsically age dependent or Poly I:C stimulation induced genes (Fig. 1a). Gene expression was compared for basal level age-related differences comparing weanling vehicle (WV) versus adult vehicle (AV) groups. Activation comparisons were done by comparing Weanling poly I:C stimulated (WPI) versus WV, Adult poly I:C stimulated (API) versus AV as well as WPI versus API. Differential gene expression volcano plots and pathway enrichment analyses (Ingenuity Pathway Analysis, IPA), from the above comparisons highlight the cellular processes that distinguish the different treatment and age groups (Supplementary Fig. 1a–d). Specific genes for further evaluation were selected based on differential expression between groups with a log2 difference of ≥ 1, p < 0.05 and base-mean ≥ 100 (Supplementary Table 1, Group 1). To evaluate the likely cell composition in the isolated microvessel fragments, we assessed expression of cell type-specific genes from the RNA seq data (Supplementary Table 2). This showed that BCEC-specific genes were highly enriched, there was moderate/low expression of pericyte markers, but very little expression of genes characteristic of astrocytes, neurons, and smooth muscle cells. This confirmed that the method used for microvessel fragment preparation resulted in highly specific BCEC enrichment, as described previously18,19,20.

Fig. 1: RNA-seq analyses and validation of target genes ex vivo.

a Sequential approach to select target genes from RNA-seq analyses on vehicle (V) or Poly I:C (PI)-treated weanling (W) or adult (A) mice (WV, WPI, AV, and API). b Identification of target genes from RNA-seq analyses of microvessel fragments from olfactory bulb (OB) and cortex (CT) regions of weanling LACV infected (LOB and LCT) and mock inoculated mice (MOB and MCT) (N = 3 for all RNA-seq samples). The default DESeq function with betaPrior = FALSE was applied, wherein a negative binomial generalized model with Wald test for significance was performed. The two-sided p-values were corrected for multiple testing using the Benjamini Hochberg method. ce Differential expression of representative genes, as validated by real-time PCR analyses on ex vivo isolated microvessel fragments from weanling mock (WM), weanling LACV (WL), adult mock (AM) and adult LACV (AL) infected mice. Gene categories: (c) intrinsic adult-specific (for Efna2 and H2q6 WM vs AM P = 0.0032 and P = 0.0001 and WL vs AL P = 0.0002 and P = 0.0356), d intrinsic weanling-specific (for Bst1 and Mmp25 WM vs AM P < 0.0001 and P = 0.1010 and WL vs AL P < 0.0001 and P = 0.4684) or (e) LACV-infection induced, adult enhanced genes (for Clec4e and Cldn1 WM vs AM P = 0.0453 and P = 0.0894 and WL vs AL 0.6490 and P = 0.5686). f Differential expression of representative genes from OB and CT microvessel fragments obtained from LACV-infected adults (AOB and ACT) and weanlings (WOB and WCT). P = 0.0043 and P = 0.0068 for Aqp1 and Ttr for WOB vs WCT comparison and P = 0.0417 and P < < 0.0001 for Aqp1 and Ttr for AOB vs ACT comparison. The data in (cf) were transformed to log2 scale for a more normal distribution and the datapoints were statistically analyzed or plotted post-transformation. Significance values were measured by one-way ANOVA followed by Tukey’s multiple comparison (ce) or two-tailed, multiple unpaired t-tests (f) (N = 5–6 for all mouse experiments, from Panel cf and individual datapoints are shown). (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

As regional differences in BBB integrity are observed following LACV infection and virus infection is first observed in the OB region in weanling mice16, we also completed an RNA-seq analysis on microvessel fragments isolated from different regions of LACV-infected (L) and mock-inoculated (M) weanling mouse brains at 3 dpi. Genes from this comparison were selected based on two differential gene expression criteria: either a difference in expression between microvessel fragments from the OB of LACV-infected mice (LOB) versus the OB of mock-infected mice (MOB), or a difference in gene expression between LOB versus microvessel fragments from CT of LACV-infected mice (LCT) (Fig. 1b). These criteria were used to select additional genes for further analysis (Supplementary Table 1, Group 2). Differential gene expression and IPA analyses from these comparisons highlight the expected enrichment of interferon and neuroinflammation following LACV infection of the OB, and differences in Gap Junction and Ephrin signaling in the OB/CT comparison (Supplementary Fig. 1e, f). Finally, in addition to the selected Group 1 and 2 genes, we also included genes known to affect BBB function and the host immune response to LACV, but not necessarily differentiated in the above RNA seq analysis (Supplementary Table 1, Groups 3 and 4, respectively).

To further confirm expression differences of the selected genes, we analyzed expression by qPCR following LACV infection. RNAs were extracted from microvessel fragments obtained from LACV-infected adult (AL) or weanling (WL) mice at 3 dpi as well as mock-inoculated weanling (WM) and adult (AM) mice, while additional microvessel fragments were extracted from OB and CT regions of LACV infected (3 dpi) weanling and adult mice. Real-time PCR analyses on ex vivo isolated microvessel fragments showed genes falling into four main categories, adult-enhanced, weanling enhanced, LACV-induced and region specific (OB-CT) (Fig. 1c–f). For example, ephrinA2 (Efna2; an angiogenic molecule) and H2q6 (belonging to MHC class I family) were expressed at higher levels in adult microvessel fragments, regardless of virus infection (Fig. 1c). Bone marrow stromal cell antigen 1 (Bst1; an immune regulator) and matrix metallopeptidase 25 (Mmp25) had higher basal levels in weanling mice (Fig. 1d). C-type lectin domain family 4, member e (Clec4e; a molecule that recognizes LACV and plays a limited role in early antiviral responses against LACV21) and claudin 1 (Cldn1; a TJ protein present in endothelial/epithelial cells) were increased by LACV infection in adult mice, but not in weanling mice (Fig. 1e). Only a few genes were differentially expressed between OB and CT microvessel fragments. Among these, two homeostasis maintenance genes aquaporin 1 (Aqp1; a passive transporter) and transthyretin (Ttr; a carrier protein) were increased in CT microvessel fragments from both weanling (WCT) and adult (ACT) mice compared to OB microvessel fragments obtained from weanling (WOB) and adult (AOB) mice. The pairwise comparison between the OB and CT microvessel fragments are shown for each age group (Fig. 1f). Thus, we identified genes by RNA seq (Fig. 1a, b) and qPCR analyses (Fig. 1c–f) that were differently expressed in microvessel fragments by age, infection, or region for further study.

Targeted siRNA screen elucidates potential LACV susceptibility or resistance factors in endothelial cells

To directly examine whether the selected genes may influence viral pathogenesis, we utilized an in vitro model of endothelial cellLACV infection. We previously found that, although infected endothelial cells are not readily detectable in vivo, they are infected in an age-dependent manner in vitro and this infection is associated with direct and bystander damage to endothelial cells17. To establish a gene-perturbation cell-based screening assay, we chose a mouse endothelioma cell line, bEnd.3, as these cells could be infected with LACV and were readily amenable to siRNA transfection. To optimize siRNA delivery, we employed lethal and nontargeting (NT) control siRNAs to test different transfection reagents and identify conditions with maximum cell killing with lethal siRNA while maintaining maximum viability with the si-NT control. This identified an optimal delivery protocol using 50 nM siRNA with the Transit TKO transfection reagent (Supplementary Fig. 2a). We then established three control conditions for the screen: a) a baseline condition for LACV infection with NT siRNAs, b) a control for known restriction factors using a combination of siRNA to Ifnar1 and Ifnar2 (si-Ifnar or si-Upreg. Control; upregulated LACV infection) and c) a control for known susceptibility factors using a combination of siRNAs to Clathrin heavy chain and Rab5a (si-CltcRab or si-Downreg. Control; downregulated LACV infection) (Supplementary Fig. 2b, c).

We then screened the 35 selected genes shown in Supplementary Table. 1 along with the three control conditions at three different phases of LACV infection on BCEC monolayers at room temperature. The first was the early phase (6 hpi; Fig. 2a), which would suggest an effect on virus entry. The second was the medial infectious phase (24 hpi; Fig. 2b), suggesting an effect on virus replication and spread through the endothelial cell culture. These kinetic phases have been similarly observed for LACV infection in a different cell line22. The cell-based assay for these first two phases used an anti-LACV antibody to measure viral fluorescence intensity on a per-cell basis. This was determined by measuring LACV sum-intensity (which is impacted by both the number of infected cells as well as the intensity of infection) and then quantitating the ratio to Hoechst area (which normalizes the intensity value to total cell number). The last, late phase assay (72 hpi; Fig. 2c) measured cell loss through LACV-induced cell death. Using these criteria, knockdown of putative restriction factors would be expected to give increased LACV intensity and reduced cell viability, while knockdown of putative susceptibility factors would lead to reduced LACV intensity and higher cell viability. We identified two clusters of genes that showed putative resistance or susceptibility phenotypes (color highlighted in Fig. 2a–c), with representative images of virus upregulation or downregulation shown in Supplementary Fig. 3.

Fig. 2: Identification of putative host restriction and susceptibility factors in LACV infection through targeted siRNA screening.
figure 2

bEnd.3 cells were transfected with 50 nM siRNA against the indicated target genes (for 72 h), along with viral upregulation control (i.e., si-Ifnar1 and Ifnar2, abbreviated as si-Upreg. Control), downregulation control (i.e., si-Cltc and Rab5a, abbreviated as si-Downreg. Control), non-targeting (si-NT, dotted line) and cell death (si-Lethal) controls where indicated. a, b Analyses of degree of infection (10 MOI of LACV) normalized to si-NT (LACV sum-intensity-to-Hoechst area ratio) at 6 (a) and 24 hpi (b). c Analyses of cell survival (nuclear count) at 72 hpi. The first bar represents the average of 2 independent gene-specific siRNA, whereas the next two bars represent siRNA#1 and #2 for each gene (mean ± SD, N = 3 each individual siRNA). Putative hit genes are color-matched across the different graphs.

Putative restriction factors identified in the primary screen included Efna2, Clec4e, Gja1 (expressed as Connexin43 protein, abbreviated as Cx43), interferon induced transmembrane protein 3 (Ifitm3, an RNA viral restriction factor), lymphocyte antigen 6 complex locus C2 (Ly6c2) and H2q6. For H2q6, there was a variation between 6 and 24 hrs in phenotype, with increased viral intensity only observed at the early time point (Fig. 2a, b). Clusters of syncytia-like aggregation and multinucleated cells were observed in H2q6 knockdown cultures, which might explain the limitation of the increased viral intensity phenotype to the early time point (white arrows, Supplementary Fig. 3a, b). Gja1 knockdown caused dissociation of the cellular monolayer (white arrow, Supplementary Fig. 3b), consistent with its established role in gap junction integrity. Putative susceptibility factors included vascular endothelial growth factor A (Vegfa), actin-cytoskeleton remodeling protein gelsolin (Gsn), claudin 2 (Cldn2), claudin 5 (Cldn5) and tight junction protein 2 (Tjp2/ZO2) (Fig. 2a–c). Thus, multiple genes were identified that either enhanced or reduced LACV in vitro infection of bEnd.3 endothelial cells.

Putative target genes control susceptibility of endothelial cells to LACV infection in different phases of viral infection

Based on the targeted siRNA screen, we chose seven putative restriction factors (Group1 genes: Bst1, Efna2, H2q6, Clec4e, Gja1, Ifitm3 and Ly6c2) for further validation in primary adult and weanling BCECs. Interestingly, knockdown of all of these genes increased susceptibility of adult primary BCECs to LACV infection (Fig. 3a), while only Ifitm3 knockdown showed a significantly increased infection level in primary weanling BCECs (Fig. 3b), supporting the premise that these genes may contribute to the increased resistance to LACV in adult BCECs.

Fig. 3: Validation of gene hits in primary culture and analyses of their effect on viral life cycle.
figure 3

a, b Adult (a) and weanling (b) primary BCECs were transfected with 50 nM siRNA for 72 h against the indicated Group1 hit genes (mainly restriction factors) and LACV infection (LACV sum-intensity-to-Hoechst area ratio) was assessed (N = 6 and individual datapoints are shown) Here, P = 0.0162, 0.0085, 0.0153, 0.0830, 0.0394, 0.0636, 0.0231 and 0.0271 in (a) and P = 0.6571, 0.9010, 0.0960, 0.1548, 0.9290, 0.0354, 0.0649 and <0.0001 in (b) for si-Bst1, si-Efna2, si-H2q6, si-Clec4e, si-Gja1, si-Ifitm3, si-Ly6c2 and si-Upreg. control vs si-NT, respectively. c LACV attachment/ entry assay in bEnd.3 cells transfected with 50 nM of the indicated gene specific and control siRNAs for 72 h and infected with 10 MOI of LACV for 24 h. d Representative images of LACV attachment/entry in Ifitm3 and Lyc2 siRNA perturbed bEnd.3 cells (green: LACV and blue: Hoechst) (N = 3 for c, d). Here, P = 0.0682, 0.0752, 0.9479, 0.3786, 0.3018, 0.0009, 0.0020 and 0.9574 in (c, left) and P = 0.0980, 0.9622, 0.6042, 0.4154, 0.9939, 0.0132, 0.0011 and 0.0294 in (c, right) for si-Bst1, si-Efna2, si-H2q6, si-Clec4e, si-Gja1, si-Ifitm3, si-Ly6c2 and si-Upreg. control vs si-NT, respectively. e Plaque assays (each dot = 1 sample) performed at 24 hpi in LACV-infected bEnd.3 cells transfected for 72 h with 50 nM of the indicated gene-specific siRNA. Here, P = 0.5075, 0.0030, 0.7566, 0.1347, 0.0120, 0.7941 and 0.0156 for si-Bst1, si-Efna2, si-H2q6, si-Clec4e, si-Gja1, si-Ifitm3 and si-Ly6c2 vs si-NT, respectively. f bEnd.3 cells were transfected with 50 nM of the indicated siRNA for 72 h, infected with 10 MOI LACV and confocal microscopy was used to image LACV infection (green), actin (red: phalloidin) and nuclei (blue: Hoechst). a, b Multiple, two-tailed paired t-tests and ce multiple, two-tailed unpaired t-tests between si-NT and the targeted genes were performed (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) and mean ± SD (3–6 samples per condition) are shown. d, f Images are representative of 25–75 independent fields. Scale bar = 100 um (white, for regular images), scale bar = 10 um (yellow, for zoomed in images).

We also tested putative susceptibility factors for validation in primary culture (Group2 genes: Cldn2, Tjp2, Cldn5, Vegfa, Mmp8, Mmp25 and Gsn). Knockdown of Mmp25, a weanling enhanced gene, led to reduced LACV intensity in both adult and weanling primary BCECs (Fig. S4), suggesting this host gene may facilitate LACV infection. However, only marginal effects were observed for the other putative susceptibility factors in primary BCECs. Since we observed a higher frequency of hit validation for Group1 genes in primary cells, we focused on genes from this group for further analysis.

In an effort to dissect mechanisms of virus inhibition, we next utilized the bEnd.3 cells to examine the stages at which perturbation of the putative restriction factors may limit virus replication. Attachment / entry assays (inducing viral binding at low temperature, see methods section) showed that two genes, Ly6c2 and Ifitm3, restricted viral entry (Fig. 3c). Compared to si-Ly6c2, si-Ifitm3 knockdown caused smaller upregulation of infected cell count as well as LACV intensity/cell (Fig. 3c, d). We then conducted plaque assays and found that perturbation of Ly6c2, Gja1 and Efna2 increased virus production at 24 hpi (Fig. 3e).

In our primary screen, we had noted that knockdown of the H2q6 gene also induced syncytia-like aggregation formation in bEnd.3 cells (Supplementary Fig. 3a, b). To investigate this further, control and H2q6-perturbed bEnd.3 cells were co-stained for both LACV and F-actin. It has been reported previously that actin cytoskeletal proteins are altered in OB BCECs following LACV infection16. We observed that LACV colocalized with the actin network in control cells, specifically at the cellular periphery and lamellipodial extensions (thin arrow, upper panel, Fig. 3f). In the H2q6 knockdown condition however, specifically in the cells having two or more nuclei (forming syncytia-like aggregation), we observed disruption of regular, filamentous actin staining and granular staining of actin and LACV located around cellular nuclei (thick arrow, bottom panel, Fig. 3f) suggesting a potential alteration of virus trafficking. Thus, in vitro validation of the putative viral restriction factors (Efna2, H2q6, Clec4e, Gja1, Ifitm3) identified from our focused siRNA screen suggests possible roles for select host genes in restricting or altering viral entry, trafficking, or viral particle release in LACV-infected endothelial cells.

Efna2 as a regulator of LACV susceptibility in vivo

One of the primary genes that significantly affected LACV infection of endothelial cells was Efna2, which is intrinsically abundant in adult BCECs. The encoded EFNA2 protein is an angiogenic factor23 that carries out signaling responses via EphrinA (EphA) class receptors24. To further examine EFNA2, we tested whether recombinant mouse EFNA2 (rec-EFNA2) protein could restrict LACV infection in weanling and adult primary BCECs. Weanling and adult primary BCECs were cultured, infected with LACV (10 multiplicities of infection; abbreviated as MOI), then maintained in EFNA2 conditioned or control medium for up to 72 h post infection. Two different EFNA2 concentrations, low (2ug/ml) or high (20 ug/ml) were used. Upon rec-EFNA2 treatment, weanling BCECs showed reduced levels of LACV infection at 24 hpi, which was significant for the high concentration of EFNA2 (Fig. 4a). The adult BCECs, already mostly resistant to LACV infection, showed partial but not significant reduction in infection. Thus, exogenously added EFNA2 can restrict in vitro LACV replication in weanling BCECs (Fig. 4a).

Fig. 4: Role of adult-specific higher expression of Efna2 in protecting BCEC from LACV infection.
figure 4

a Effect of recombinant EFNA2 (2 ug/ml to 20 ug/ml) on LACV infection levels in weanling and adult BCECs at 24 and 72 hpi (N = 4–12 individual samples, data collected based on 25 images for each sample, P = 0.1654 and 0.0475 for 2 ug/ml and 20 ug/ml, compared to control, in weanling BCECs and P = 0.1068 and 0.0846 for 2 ug/ml and 20 ug/ml, compared to control, in adult BCECs). b Adult Efna2−/− (m), Efna2+/− (m) and WT mice were infected with 105 PFU LACV (IP) and the percentage of neurologic mice are shown. See Suppl. Table 3 for mixed Efna3/5 genotypes of Efna2−/− (m) and Efna2+/− (m) mice (N = 22 WT or Efna2+/− (m) mice and N = 18 Efna2−/− (m) mice, P = 0.0105). c LACV RNA levels in the brains of infected mice at 8–22 dpi (NC: nonclinical mice and C: clinical mice where N = 18 WT or Efna2+/− (m) NC mice, N = 2 WT or Efna2+/− (m) C mice, N = 10 Efna2−/− (m) NC mice and N = 8 Efna2−/− (m) C mice). Here, P = 0.0008 for both WT/ Efna2+/− (m) C and Efna2−/− (m) C, compared to WT/ Efna2+/− (m) NC control. d Vascular leakage as assessed by the measurement of viral RNA in LACV-infected Efna2−/− (m) and WT mice brains at 3 dpi (N = 8 mice brains regarding each condition analyzed). e Imaging to detect vascular leakage in brain slices from LACV-infected WT and Efna2−/− (m) mice at 3 dpi (arrow, FluoSphere beads: red, Hoechst: blue). Thick arrows represent localization of FluoSphere beads at CNS periphery in WT mice whereas the thin arrows represent leakage of FluoSphere beads into the brain parenchyma in Efna2−/− (m) mouse (1 out of 4 mice showed leakage, which is represented here). (a, one way ANOVA followed by post-hoc Dunnett’s test, b, Mantel-Cox log rank test and c, d multiple unpaired, two-tailed t-tests, *P < 0.05, **P < 0.01 and ***P < 0.001).Scale bar = 100 um.

To examine whether Efna2 has a role in restricting LACV infection of the CNS in vivo, we analyzed the development of neurological disease in normally resistant adult mice deficient in Efna family members. We initially tested >6weeks old mice that had mixed deficiency genotypes for Efna2, Efna3 and Efna5 (Efna2−/− mixed; abbreviated as Efna2−/− (m)), as detailed in Supplementary Table 3. All mice were distributed in two groups: homozygous for Efna2 deficiency with mixed +/+, +/− or −/− genotypes for Efna3 and Efna5 (Efna2−/− (m) or heterozygous for Efna2 deficiency with mixed +/+, +/− or −/− genotypes for Efna3 and Efna5 (Efna2+/− (m)) as well as wildtype mice with Efna2+/+3+/+5+/+ genotype (wildtype, abbreviated as WT). Following inoculation of 105 PFU/mouse, approximately 50% of Efna2−/− (m) mice developed neurologic disease, regardless of their Efna3 or Efna5 genotype (Supplementary Table 3). In contrast, most of the WT or Efna2+/− (m) mice (~91%) did not show any signs of neurological disease (Fig. 4b). We also observed high levels of virus in the Efna2−/− (m) mice with clinical (C) neurological disease versus non-clinical (NC) mice (Fig. 4c). Thus, Efna2 appears to have an inhibitory role in LACV pathogenesis in vivo (Fig. 4b, c).

To determine if Efna2 deficiency affects BBB permeability and LACV neuroinvasion, we examined mice at 3 dpi, four days prior to the onset of clinical signs. LACV RNA was detected in approximately ½ of the Efna2 −/− (m) mice, but in only one mouse from the WT and Efna2+/− (m) group (Fig. 4d). These LACV-infected Efna2−/− (m) mice were also injected with ~100 nm red FluoSphere beads (virus-sized particle; red staining) at 3 dpi to monitor vascular leakage. Fluorospheres were primarily detected within CNS blood vessels in WT mice (thick white arrows). However, in one of the four Efna2−/− mice, fluorospheres were detected within the brain parenchyma (thin white arrows, Fig. 4e) suggesting early vascular leakage. Thus, the 25–50% increased disease in Efna2−/− (m) mice correlated with increased incidence of early viral RNA detection in the CNS and detection of vascular leakage in a subset of these mice. These data indicate that Efna2 may be an important restriction factor in adult mice to prevent LACV neuroinvasion.

To clarify whether Efna2 itself was the primary mediator of viral restriction, we compared LACV susceptibility in adult (> 6 weeks old) WT and Efna2−/−3+/+5+/+ mice (Efna2 single knockout (KO); abbreviated as Efna2−/−(s)). As expected, the adult WT mice were not susceptible to a 105 PFU/mouse LACV dose (~96% non-neurologic). In contrast, approximately 38% of adult Efna2−/− (s) mice developed neurological disease in response to LACV infection (Fig. 5a). Furthermore, microvessel fragments isolated from Efna2−/−(s) mice had higher levels of LACV infection compared to WT at 24 and 48 hpi (Fig. 5b, d) and reduced survival at 72 and 96 hpi (Fig. 5c). We also examined vascular leakage in vivo using fluorospheres in WT and Efna2−/− (s) mice using a slightly later timepoint of 5 dpi. WT adult mice showed no vascular leakage, while Efna2−/− (s) mice had consistent detection of 1–3 fluorescent bead foci in the brain parenchyma (Fig. 5e, 5 out of 7 mice). Thus, Efna2−/− (s) mice were more susceptible to LACV-induced neurological disease, which correlated with an increase in vascular leakage in the CNS prior to disease onset. This also correlated with increased virus infection and decreased cell survival in Efna2−/− (s) BCECs compared to WT BCECs (Fig. 5b, c). Collectively, these data indicate an important role for Efna2 in BBB integrity during LACV infection.

Fig. 5: Loss of Efna2 confers susceptibility in adult LACV-resistant mice.
figure 5

a Comparison of LACV-induced (105 PFU/mouse dose (IP)) neurologic disease in adult WT and Efna2−/− mice. (*P < 0.05, ****P < 0.0001, Mantel-Cox log rank test, N = 28 WT mice and N = 43 Efna2−/− mice, P = 0.0013). b Comparative infection of BCECs after LACV infection (10 MOI) at 24 and 48 hpi on WT and Efna2−/− primary BCECs (N = 33 and N = 18 for WT and Efna2−/− BCECs, mean ± SD is shown, P < 0.0001 and P = 0.0041 for 24 and 48 hpi, respectively). c Comparative cell viability (validated nuclear count) of BCECs after LACV infection (10 MOI) at 72 and 96 hpi on WT and Efna2−/− primary BCECs (N = 24 and = 12-18 for WT and Efna2−/− BCECs, mean ± SD is shown, P < 0.0001 and P = 0.0304 for 72 and 96 hpi, respectively). All individual datapoints are shown and statistical significance analyzed by multiple, two-tailed unpaired t-tests, *P < 0.05, **P < 0.01, and ****P < 0.0001). d Representative images at 24 and 48 hpi comparing WT and Efna2−/− BCECs (Hoechst: Blue and LACV: green). Scale bar = 100 um (images are representative of 25 individual images/ sample). e One representative WT brain section (without any vascular leakage) is shown along with 5 Efna2−/− brain sections demonstrating vascular leakage (Hoechst: blue and FluoSphere beads: red and scale bar = 100 um) (N = 5 WT brains and N = 7 Efna2−/− brains, of which 5 brains showed probable foci of vascular leakage). The number of foci/ brains was compared using an unpaired t-test (P = 0.0195).

Small molecule-based activation of Cx43 protein delays LACV-induced neurological endpoint in weanling animals

Since knockdown of Gja1 (which expresses the Cx43 protein) impacted LACV infection in vitro, we hypothesized that activating or upregulating Cx43 might reduce virus infection and pathogenesis. 4-Phenylbutyric acid (4-PBA) is a known activator and channel formation enhancer for several connexins, including Cx43. We infected bEnd.3 cells with LACV + /− 4-PBA and immunostained for LACV and Cx43 at different time points. 4-PBA treatment significantly reduced LACV infection and increased host cell survival at 96 hpi (Fig. 6a). Using confocal microscopy, we observed that the reduction of LACV infection (LACV: green) at 96 hpi correlated with enhancement of Cx43 puncta formation (Cx43: red). The increase in Cx43 puncta formation (white arrows; denoted by granular or laminar Cx43 staining) or localization toward the cell surface was 4-PBA dose dependent (Fig. 6b)25.As observed in previous studies25, the upregulation of Cx43 protein level was confirmed by western blot (Fig. 6c). Thus, 4-PBA-induced upregulation in Cx43 protein levels or puncta formation correlated with inhibited of LACV infection in vitro.

Fig. 6: 4-PBA induced alteration of Cx43 (expressed by Gja1 mRNA) alters LACV induced vascular leakage and viral entry into weanling mice brain.
figure 6

a, b LACV-infected (10 MOI) bEnd.3 cells, were treated with 4-PBA (0–10 mM) and at different hpi, imaged to measure (a) viral intensity or virus-induced cell depletion (N = 4 samples, data collected based on 25 images obtained from each sample and mean with SD is shown) or (b) localization of Cx43 (red), LACV (green) and Hoechst (blue) (representative of ~12–25 images obtained each condition, P = 0.1142, < 0.0001 and 0.0097 from PBA 2.5, 5and 10 mM, compared to PBA 0 mM). White arrows represent punctate staining of Cx43. c Western blot and densitometric analyses of Cx43 protein levels in 4-PBA treated bEnd.3 cells. Gapdh is a loading control and normalization factor for quantitation (N = 3 each condition and mean with SD is shown, P = 0.7647, 0.0043 and 0.4589 for PBA 2.5, 5 and 10 mM those are compared to PBA 0 mM). d Weanling mice, infected with 2000 PFU of LACV, were treated with vehicle (LV) or 500 mg/kg-day 4-PBA (LP) until 5 dpi and assessed for neurologic endpoint (N = 9 mice for each condition, P = 0.0074). e Effect of 4-PBA treatment on the correlation between LACV and Gja1 expression in mouse brains at 3 dpi (N = 9 LV and N = 11 LP brain RNA samples obtained from mice, P = 0.0311 and 0.0147 for LACV and Gja1 comparison). f Effect of 4-PBA on entry and localization of LACV as assessed by confocal imaging of mouse brain slices at 4 dpi (red: ZO1, green: LACV, blue: Hoechst/nuclei). (a) two-way ANOVA followed by Dunnett’s test and all groups were compared with vehicle for same time pi, (b) one-way ANOVA followed by Dunnett’s test, individual points represent separate imaging fields, (c) multiple, two-tailed unpaired t-tests, d Mantel-Cox log rank test and e, two-tailed unpaired t-test where *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 and N = 3–4 for each experiment. Scale bar = 100 um (white, for regular images), scale bar = 10 um (yellow, for zoomed in images).

To examine if 4-PBA could inhibit LACV-induced neurological disease, LACV infected weanling mice (~21–23 days old) were treated daily with 500 mg/kg 4-PBA from 0 to 5 dpi (IP injection, LP). LACV infected and vehicle treated mice (LV) were analyzed in parallel. The LV mice exhibited neurological disease between 6 and 8 dpi. In contrast, LP mice had delayed development of neurological disease (Fig. 6d). At the neurological endpoint, virus levels and Gja1 mRNA expression levels were similar in vehicle and 4-PBA treated groups (Supplementary Fig. 5). However, earlier analysis at 3 dpi, when virus first invades the CNS, demonstrated that LP animals had reduced virus levels, which correlated with an increase in Gja1 mRNA expression (Fig. 6e). At 4 dpi, brains were isolated from LV and LP mice and co-stained for the endothelial marker ZO1 and LACV. LV mice showed persistent and prominent presence of LACV staining in the OB and OT region of the brain (Fig. 6f; upper panels). In contrast, 4-PBA-treated mice had either no detectable LACV (3 of 4 mice) or much more limited viral staining (1 out of 4 mice, (Fig. 6f; lower panels)) compared to vehicle controls. Higher magnification images showed prominent LACV infection in brain parenchyma in a highly vascularized area in LV mice, whereas LACV infection was restricted primarily to the vascular lumen at 4 dpi in LP mice (Fig. 6f, right side panels). Notably, virus particles were restricted inside the vessels without establishing a sufficient leakage inside brain parenchyma at 4 dpi. Thus, 4-PBA treatment induced upregulation of Cx43 and could partially restrict viral infection and dissemination in the OB/OT region at the early stage of LACV infection.

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