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. 2024 Jan-Dec;16(1):2428425.
doi: 10.1080/19490976.2024.2428425. Epub 2024 Nov 20.

Rotavirus rewires host cell metabolic pathways toward glutamine catabolism for effective virus infection

Affiliations

Rotavirus rewires host cell metabolic pathways toward glutamine catabolism for effective virus infection

Suvrotoa Mitra et al. Gut Microbes. 2024 Jan-Dec.

Abstract

Rotavirus (RV) accounts for 19.11% of global diarrheal deaths. Though GAVI assisted vaccine introduction has curtailed RV induced mortality, factors like RV strain diversity, differential infantile gut microbiome, malnutrition, interference from maternal antibodies and other administered vaccines, etc. often compromise vaccine efficacy. Herein emerges the need of antivirals which can be administered adjunct to vaccination to curb the socio-economic burden stemming from frequent RV infection. Cognisance of pathogen-perturbed host cellular physiology has revolutionized translational research and aided precision-based therapy, particularly for viruses, with no metabolic machinery of their own. To date there has been limited exploration of the host cellular metabolome in context of RV infection. In this study, we explored the endometabolomic landscape of human intestinal epithelial cells (HT-29) on RV-SA11 infection. Significant alteration of host cellular metabolic pathways like the nucleotide biosynthesis pathway, alanine, aspartate and glutamate metabolism pathway, the host citric acid cycle was observed in RV-SA11 infection scenario. Detailed study further revealed that RV replication is exclusively dependent on glutamine metabolism for their propagation in host cells. Glutamine metabolism generates glutamate, aspartate, and asparagine which facilitates virus infection. Abrogation of aspartate biogenesis from glutamine by use of Aminooxyacetic acid (AOAA), significantly curbed RV-SA11 infection in-vitro and in-vivo. Overall, the study improves our understanding of host-rotavirus interactome and recognizes host glutamine metabolism pathway as a suitable target for effective therapeutic intervention against RV infection.

Keywords: Rotavirus; antiviral; aspartate aminotransferase; glutamine metabolism; metabolomics.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Profiling of host cellular metabolome during RV infection in HT-29 cells. Metabolomic analyses was performed in n = 3 samples each of mock and RV-SA11 infected HT-29 cells. (a) PCA (Principal component analysis) of mock (red) and RV-SA11 (M.O.I. = 3) infected HT-29 cells (blue) at (i) 6 h (ii) 12 h post RV-SA11 infection revealed differential clustering of mock and infected cells based on altered regulation of metabolites. (b) Intersecting venn diagram depicting 131 and 227 altered metabolites at 6 (red) and 12 h.pi. (blue) respectively in RV-SA11 infected cellular lysates compared to mock, with 35 common metabolites (p < 0.2) differentially altered at both 6 h.p.i. and 12 h.p.i. (c) Heatmap analyses of mock and RV infected HT-29 cells at (i) 6 h.p.i. And (ii) 12 h.p.i. depicted differentially regulated metabolites at p < 0.2. Blue indicates downregulation and red indicates upregulation. (d) Bubble plots representing the most significantly altered cellular metabolic pathways during RV infection. Pathway analysis was done by mapping the differentially regulated metabolites (p < 0.2) to host cellular biochemical pathways using their HMDB/KEGG I.D.s and screening the hits against the KEGG pathway database in the Metaboanalyst software. The top 5 pathways with impact score ≥ 0 and false discovery rate (FDR) <0.7 at (i) 6 h.p.i. and (ii) 12 h.p.i. are represented in adjoining table. Circle size is indicative of pathway impact (bigger circle; greater impact), while, color/gradient of the circle (from red to yellow) indicates the level of statistical significance. (deeper red indicates higher-log10(p) value and increased significance).
Figure 2.
Figure 2.
Rotavirus replication in human intestinal epithelial cells is dependent on host cellular glutamine. (a) Rates of consumption of glutamine from cellular media of mock and RV-SA11 infected HT-29 cells were determined in Relative Luminescence Units (RLU) using the Promega Glutamine/Glutamate Glo assay kit at increasing timepoints. Increased uptake of glutamine was evident in RV-SA11 infected cells at 6 and 12 h.p.i. compared to the mock HT-29 cells (p ≤ 0.05; F = 16.82, df = 1) (b) (i) HT-29 cell survivality was measured through MTT assay after incubating cells for 24 h in varied concentrations of glucose and glutamine. (ii) Expression of RV-VP6 and RV-NSP5 was determined through immunoblotting using specific antibodies, in cellular lysates of RV-SA11 infected HT-29 cells (12 h.p.i), in DMEM media with varied concentrations of glucose and glutamine. The blots were normalized against the internal loading control, β-actin. Absence of glutamine from media during RV-SA11 infection substantially decreased VP6 and NSP5 protein synthesis. (c) (i) Relative fold changes in transcript levels of RV-VP6 was quantified using SYBR-Green based qRT-PCR assay after normalization with 18S internal control. Significant increase in VP6 transcript levels was observed on increasing glutamine concentration in infection media above 1 mM (p ≤ 0.05, F = 53.27, df = 5). Each bar in the bar graph is a representation of the mean ± SD of a minimum of three replicates. (ii) RV-VP6 and RV-NSP5 protein levels were analyzed by western blot analysis. Immunoblotting results demonstrated similar trend of increase in RV-SA11 VP6 and NSP5 with increasing gradients of glutamine in infection media. (iii) Total number of viroplasm positive cells (HT-29) in increasing concentrations of glutamine (0, 0.25, 1, 2, 3 and 4 mM) in special DMEM media containing 5 mM glucose was also determined through confocal microscopy. Total number of viroplasm positive cells increased when glutamine concentration in infection media was 1 mM or more (p ≤ 0.05, F = 45.32, df = 5). (d) Disrupting glutamine utilisation by treating RV -infected HT-29 cells with the glutamase inhibitor, L-DON, in a dose dependent manner, was found to drastically reduce RV-VP6 protein levels. Test of significance used was two-way ANOVA for glutamine uptake assay and one-way ANOVA for the rest followed by post-hoc Tukey test comparing all pairs of columns (ns denotes p value not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Figure 3.
Figure 3.
RV infection in HT-29 cells is associated with altered regulation of different enzymes of glutamine metabolism pathway; functional inhibition of which decreases rotavirus replication. (a) Schematic diagram of the glutamine metabolism pathway in host cells. (b) Protein level expression of enzymes involved in glutamine metabolism: glutaminase (GLS: 65 kda), glutamate dehydrogenase (GLUD: 54 kda)), glutamate oxaloacetate transaminase 1 & 2 (GOT1 and GOT2: 41 kda) and asparagine synthetase (ASNS: 64 kda) was quantified in whole cell lysates of HT-29 cells at 6, 12, 18 and 24 h.p.i. and compared to the un-infected controls. The protein expression levels were normalized with respect to the internal loading control, β-actin. RV-NSP5 expression was also assessed as a marker of virus infection. Compared to the mock-infected HT-29 cells, increased protein expression was observed for GLS, GOT2 and ASNS, at early time-points. (C-E) RV- SA11 infected cells were treated with the increasing concentrations of functional inhibitors of (c) GLS (CB-839 (10 - 20 μM) (D) GLUD (R-162 (20,30 μM) and (E) AOAA (0,150 and 300 μM) 1 h post infection and maintained for 12 h in DMEM containing 5 mM glucose and 2 mM glutamine. Viral RNA transcripts (VP6) were quantified in infected and drug-treated cells after normalization with housekeeping gene, 18S. Each bar in the bar graph represents mean ± SD of a minimum of three replicates. Significant decrease in viral RNA was evident on treatment with inhibitors CB-839 (p ≤ 0.05, F = 18.82, df = 2) and AOAA (p ≤ 0.05, F = 45.16, df = 2). Whole cell lysates were also used for Western blotting to determine changes in viral proteins, VP6 and NSP5 normalized against loading control, β-actin. Reduced levels of RV-VP6 and NSP5 proteins were evident in cells treated with CB-839 and AOAA. For R162 treatment, the decline in RV-SA11 protein or transcripts were not as significant as observed on treatment with CB-839 or AOAA. (f) HT-29 cells were transfected with negative control siRNA (150 nM) or siASNS (75, 100 and 150 nM) and subsequently infected with RV-SA11 followed by incubation till 12 h.p.i. The relative transcript expression level of VP6 RNA was quantified after total cellular RNA isolation and qRT-PCR. Western blotting was used to visualize RV-SA11 VP6 and NSP5 proteins. siASNS transfection at 100 and 150 nM concentration significantly reduced RV-SA11 RNA (p < 0.05, F = 18.78, df = 3) and protein synthesis. One way ANOVA followed by post-hoc Tukey’s test comparing all pair of columns was used to determine level of significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Figure 4.
Figure 4.
Aspartate and asparagine supplementation to glutamine depleted/inhibitor treated HT-29 cells with RV-SA11 infection substantially rescues virus replication. (a-e) HT-29 cells were infected with RV-SA11 (M.O.I. 3) and incubated for 12 h in media containing 5 mM glucose and 2 mM glutamine; 5 mM glucose with no glutamine and 5 mM glucose with 2 and 4 mM each of glutamate, alpha-ketoglutarate, aspartate, asparagine and oxaloacetate. Expression levels of RV-VP6 and NSP5 proteins were determined by immunoblotting with specific antibodies and the quantified relative fold change (with respect to the first lane) was calculated after normalization against the housekeeping protein, β-actin. The low level of RV proteins in glutamine depleted media was mostly restored on addition of 2 mM and 4 mM of aspartate or asparagine to the media (DMEM with 5 mM glucose). (f) Intermediates of the glutaminolysis pathway (4 mM) were added individually or in combination in RV infected cells maintained in glutamine depleted media and changes in expression of RV proteins was assessed by immunoblotting. Asparagine either solely or in combination with other intermediates like alpha-ketoglutarate, glutamate or oxaloacetate was able to maximally restore RV infection in glutamine depleted media. (G-I) HT-29 cells infected with RV-SA11 were treated with inhibitors/siRNA targeting GLS, GOT1/2 and ASNS. Rescue experiments were performed by addition of intermediates downstream to the step catalyzed by the enzymes targeted with their functional inhibitors and viral protein levels checked through immunoblotting RV-VP6 and NSP5 protein levels were found mostly restored on addition of aspartate or asparagine, either singly or in combination with other supplements.
Figure 5.
Figure 5.
Aminooxyacetic acid, a known inhibitor of aspartate transaminases, displays potent anti-rotaviral efficacy in-vivo in BALB/C suckling mice. (a) workflow of RV-SA11 infection and treatment in BALB/C mice. (a) Four-day old suckling BALB/C mice pups were chosen for the experiment. For each experimental setup, n = 5 mice pups were taken. Mice were infected with RV-SA11 and AOAA was administered after 12 h of virus inoculation as described in methods. Expression of RV-VP6 was determined in intestinal tissue homogenates of RV-SA11 infected and drug treated (AOAA at 5 mg/kg and 10 mg/kg) mice by Western blotting using antibody specific to VP6. Expression of VP6 protein was normalized to the housekeeping gene, β-actin. (b) Total RNA was extracted from intestinal tissue homogenates of infected and treated mice and the levels of viral RNA, VP6 and NSP4 were quantified with respect to housekeeping gene, 18S. Each bar in the plotted graph is a representation of mean ± SD fold change of at least three replicates. Significant decline in VP6 (p ≤ 0.05, F = 28.41, df = 2) and NSP4 transcripts (p ≤ 0.05, F = 10.39, df = 3) in mice intestinal homogenates was observed on treatment with AOAA. (C) Stool samples were collected from RV-SA11 infected and AOAA (10 mg/kg/day) treated suckling mice and total virus particles were quantified in the stool samples at 72 h.p.i. Total virus particles in stool of AOAA treated RV infected suckling pups at the end of 72 h was significantly less than those with RV-SA11 infection (p ≤ 0.05, t = 72.35, df = 4). (d) Intestinal tissue homogenates were prepared from untreated and AOAA treated mice pups with and without RV-SA11 infection and aspartate aminotransferase activity was measured from the homogenates using the colorimetric aspartate aminotransferase assay kit from Abcam. AOAA treatment significantly reduced the intestinal aspartate aminotransferase enzyme activity in the mice pups, which was otherwise highly elevated in virus infection (p ≤ 0.05, F = 38.71, df = 3) (e) HE staining of small intestinal tissue section of 4-day-old mice uninfected, infected with RV-SA11, infected and treated with AOAA (10 mg/kg/day) were done to identify changes in tissue morphology. Restoration of damaged intestinal villi morphology was observed in AOAA (10 mg/kg/day) treated RV infected mice. Scale bars taken were 30 µm. Unpaired t-test (for comparing the results of viral titre determination in mice stool samples) or the one-way ANOVA followed by post-hoc Tukey’s test comparing all pair of columns, was used to determine level of significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

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