Anopheles gambiae heat shock protein cognate 70B impedes o'nyong-nyong virus replication

Abstract

Background: Phylogenetic and functional analysis was conducted on an Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1alpha (EF-1alpha) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1alpha and the agglutinin functions in ONNV replication in An. gambiae.

Results: Among these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of beta-galactosidase (dsbeta-gal). ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with dsbeta-gal treated mosquitoes.

Conclusion: These results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector, An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.

Figure 1

Phylogenetic tree and multiple sequence alignment of the HSP70 family from An. gambiae and D. melanogaster. The numbers for interior branches represent bootstrap values. The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position. Accession numbers of the nucleotide and amino acid sequences used: An. gambiae: HSP70A1, ENSANGG00000001248; HSP70A2, ENSANGG00000022650; HSC70A, ENSANGG00000019768; HSC70B, ENSANGG00000017398; HSC70C, ENSANGG00000016503; HSC70D, ENSANGG00000010404; HSC70E, ENSANGG00000012804; ENSANGG00000017748. D. melanogaster: Hsp70Bb, CG31359; Hsp70Bbb, CG5834; Hsp70Bc, CG6489; Hsp70Bb, CG31359; Hsp70Ab, CG18743; Hsp70Aa, CG31366; Hsp68, CG5436; Hsc70-2, CG7756; Hsc70-1, CG8937; Hsc70-4, CG4264; Hsc70-5, CG8542; Hsc70-3, CG4147; CG7182; Hsc70Cb, CG6603; CG2918.

Figure 2

Multiple sequence alignment of 5' end of coding and non-coding regions of HSC70 family. The arrows and red characters represent the highly polymorphic sites for the qRT-PCR primers. The bold characters represent the HSC70B gene and the DNA template for the dsRNA of HSC70B. The asterisks denote the conserved sequences among the HSC70 gene family. Accession numbers of the nucleotide and amino acid sequences used: An. gambiae: HSC70A, ENSANGG00000019768; HSC70B, ENSANGG00000017398; HSC70C, ENSANGG00000016503; HSC70D, ENSANGG00000010404; HSC70E, ENSANGG00000012804.

Figure 3

RNA interference efficiency. (A) Expression levels were measured by RT-PCR before (Ctr) and 6 days after the respective dsRNA (RNAi) injection based on agglutinin, HSC70B, and EF-1α and β-galactosidase (dsβ-gal) as a control. Primers for RT-PCR were designed from agglutinin, HSC70B, and EF-1α as well as ribosomal protein gene S7 (RpS7). The expression of RpS7 (23 cycles) served as a loading control. (B) The ribosomal protein gene S4 (RpS4) and HSC70B transcript levels (mean ± SD) were measured by quantitative RT-PCR at 6 days after ONNV-eGFP and dsHSC70B and dsβ-gal injections with 3 biological replicates. Primers for qRT-PCR were designed from RpS4 and HSC70B (Table 1). The transcript levels of the loading control (RpS4) did not show significant differences between dsHSC70B and dsβ-gal treatments. However, the HSC70B transcript level in An. gambiae with dsHSC70B injection show an average 58% reduction of transcript levels compared to that of the control mosquitoes with dsβ-gal treatment (Student's paired t-test, P = 0.0047).

Figure 4

ONNV titers (mean ± SD) in mosquitoes coinjected with ONNV and the respective dsRNA, including dsβ-gal, ds-agglutinin, dsHSC70B, and dsEF-1α. ONNV titers in mosquitoes coinjected with ONNV and dsHSC70B had a statistically significant increase compared to that of coinjection of ONNV and dsβ-gal at 6 days p.i. (unpaired t-test, P = 0.00045). However, ONNV titers of the dsRNAs of the other genes were not significantly different at 6 days p.i. Each data point was generated by five independent mosquitoes which were co-injected by dsRNA and ONNV.

Figure 5

The phenotype of An. gambiae in response to coinjection of ONNV-eGFP and dsHSC70B. (A) Characterization of viral transcripts of ONNV-eGFP after coinjection with dsβ-gal into adult female An. gambiae (4arr strain). Ctr, 3 and 6 represent transcript profile of the recombinant ONNV with enhanced green fluorescent protein (ONNV-eGFP) before, at 3 and 6 days p.i., respectively. Primers for RT-PCR were designed from the ONNV structural E1 gene (E1) and non-structural protein 1 gene (nsP1) (Table 1). The PCR products of the E1 and nsP1 primer pairs show the expected sizes, 431 and 440 base pairs, respectively (Table 1), indicating the correct expression of ONNV-eGFP in An. gambiae. (B) The strong expression of GFP in head, thorax, and abdomen of An. gambiae at 6 days after coinjected with dsHSC70B and ONNV-eGFP. (C) The relatively weak expression of GFP in the thorax of An. gambiae at 6 days after coinjected with dsβ-gal and ONNV-eGFP. The arrows indicate the tissues of An. gambiae with GFP expression.

Figure 6

The relative percentages (mean ± SD) of surviving mosquitoes after the coinjection of ONNV and the respective dsRNA targeting HSC70B and β-gal (black and gray, respectively). The number of mosquitoes injected represented 100%. Asterisks indicate significant differences in survival (unpaired t-test, P ≤ 0.019). N = 6 groups of 15 individuals for each data point.