Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease

A diarylamine derived from anthranilic acid inhibits ZIKV replication

Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3Hel) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3Hel

RRV-YF is attenuated <i>in vivo</i>.

<p>Disease scores (<b>A</b>) and weights (<b>B</b>) of twenty day old C57BL/6 wt mice injected subcutaneously with 10⁴ pfu of RRV-wt or RRV-YF or mock-infected with PBS. <b>C.</b> Representative histology images (hematoxylin-eosin staining) of quadriceps at 10 days p.i. Scale bar: 100 μm Virus titres from tail blood (<b>D</b>), spleen, quadricep, and ankle (<b>E</b>) at different time points after infection, determined by plaque assay. Data are presented as mean ± SEM of 3–4 mice per group. Statistical significance for the difference between RRV-wt- and RRV-YF-infected mice was determined using two-way ANOVA with Bonferroni post-test (B, D, and E) or non-parametric Mann–Whitney test (A). 0.05> p < 0.01; ** 0.01> p < 0.001; *** p < 0.001.</p

SFV-YF fails to activate AKT and does not increase glucose metabolism.

<p>Concentrations (mM) of <b>A.</b> lactate in the media, and <b>B.</b> AMP and UMP and <b>C.</b> glycerophosphocholine in the cells of samples mock-, WT SFV- or SFV-YF-infected at MOI 1 (8 hpi). Concentrations (in mM) of lactate and glucose in the media (<b>D</b>) and in the cells (<b>E</b>) of samples mock-, WT SFV- or SFV-YF-infected at MOI 5 (8 hpi). Six samples per group were analysed. Data are presented as means ± SD. SFV release of new virus particles upon 8h treatment with Wortmannin (2 μM) and MK-2206 (7 μM) in differentiated SH-SY5Y (<b>F</b>) and MK-2206 (7 μM) in primary rat cortical neurons (<b>G</b>). Data are presented as means ± SEM. Statistics as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006835#ppat.1006835.g001" target="_blank">Fig 1</a>. ANOVA was performed when comparisons included more than two groups.</p

The YXXM motif in SFV nsP3 mediates PI3K/AKT hyperactivation.

<p><b>A.</b> AKT activation in BHK cells infected at MOI 10 as indicated and lysed at 8 hpi, together with densitometry of phosphorylated AKT (S473), normalised to total AKT signals and relative to mock-infected cells (mean of three independent experiments ± SEM). <b>B.</b> Immunofluorescence showing activation of AKT (S473, green) in MEFs transfected for 24 h with the indicated DNA plasmids (red: FLAG-nsP3, blue: nuclei). Representative epifluorescence images are shown. Scale bar: 20 μm. Co-immunoprecipitations showing the interaction between EYFP-p85−α (<b>C</b> and <b>E</b>) or EYFP-p85−β (<b>D</b>) in HEK293T cells transfected with the indicated constructs for 24 h. EYFP-tagged p85 constructs were immunoprecipitated with an anti-GFP antibody. BAP: biotin acceptor peptide, Myr-Pal: myristoylation and palmitoylation. <b>F.</b> Interaction of endogenous p85 and viral nsP3 in lysates of BHK cells infected at MOI 10 for 8 h, after immunoprecipitation with an anti-p85 antibody. Growth curves of SFV-wt and SFV-YF in BHK cells infected at MOI 0.1 (<b>G</b>) or 10 (<b>H</b>) for the indicated times. Virus titres at each time point were determined by plaque assay. Data show the mean ± SEM of three experiments.</p