Mutations in the E2 glycoprotein and the 3\u27 untranslated region enhance chikungunya virus virulence in mice

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies of humans and experimentally infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected Rag1 -/- mice at day 28. When inoculated into naive wild-type (WT) mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity than the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3' untranslated region (3'-UTR). The introduction of these changes into the parental virus conferred enhanced virulence in mice, although primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3'- UTR deletion. Finally, studies with Irf3/Irf7 -/- and Ifnar1 -/- mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of interferon regulatory factor 3 (IRF3)-, IRF7-, and IFNAR1-mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection

A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

<div><p>During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMV_TRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.</p></div

Species-specific MARCO-alphavirus interactions dictate chikungunya virus viremia

Summary: Arboviruses are public health threats that cause explosive outbreaks. Major determinants of arbovirus transmission, geographic spread, and pathogenesis are the magnitude and duration of viremia in vertebrate hosts. Previously, we determined that multiple alphaviruses are cleared efficiently from murine circulation by the scavenger receptor MARCO (Macrophage receptor with collagenous structure). Here, we define biochemical features on chikungunya (CHIKV), o’nyong ’nyong (ONNV), and Ross River (RRV) viruses required for MARCO-dependent clearance in vivo. In vitro, MARCO expression promotes binding and internalization of CHIKV, ONNV, and RRV via the scavenger receptor cysteine-rich (SRCR) domain. Furthermore, we observe species-specific effects of the MARCO SRCR domain on CHIKV internalization, where those from known amplification hosts fail to promote CHIKV internalization. Consistent with this observation, CHIKV is inefficiently cleared from the circulation of rhesus macaques in contrast with mice. These findings suggest a role for MARCO in determining whether a vertebrate serves as an amplification or dead-end host following CHIKV infection

The PKR pathway is activated in HuPKR F489S cells after infection with VacVΔE3L+HCMV_TRS1.

<p>HeLa PKR KO cells with stably integrated HuPKR or HuPKR F489S were induced with doxycycline to express PKR and 24 hours later mock-infected or infected at an MOI of 3 with the indicated viruses. At 6 hpi, cells were lysed and levels of PKR-P, total PKR, eIF2α-P, total eIF2α and actin were evaluated by western blotting.</p

Species-specific differences in primate CMV TRS1 PKR antagonism map to a single amino acid.

<p>(A) Schematic representation of the SEAP assay. Transfection of PKR leads to decreased activity of a co-transfected reporter construct expressing SEAP. This PKR-driven repression can be counteracted by co-transfection of a functional TRS1 antagonist, resulting in a rescue of SEAP activity. (B) The SEAP assay recapitulates species-specific differences in HuPKR antagonism by TRS1 alleles. HeLa PKR KO cells were co-transfected with a SEAP reporter plasmid along with either a control vector or HuPKR and the indicated TRS1 alleles or a vector control. At 48 h post-transfection, SEAP activity in the medium was measured (mean ± s.d., n = 2). Data are representative of three independent experiments. (C) A single amino acid change, F489S, confers resistance to HCMV_TRS1. Point mutants were generated in HuPKR to introduce the six AgmPKR-specific residues that differ between HuPKR and AgmPKR within the region spanning codons 475 to 520, shown in the alignment. The ability of the point mutants to antagonize HuPKR was evaluated as described in (B) (mean ± s.d., n = 2). Data are representative of three independent experiments.</p

The F489S mutation eliminates HCMV_TRS1 binding to PKR.

<p>HeLa PKR KO cells were co-transfected with WT HuPKR or HuPKR F489S and either His-tagged HCMV_TRS1, AgmCMV_TRS1, SmCMV_TRS1 or EGFP. At 48h post transfection, lysates were prepared and incubated with nickel-agarose beads. Cell lysates and bound proteins were analyzed by western blotting, probing for His-tagged TRS1 proteins and for PKR. Data are representative of three independent experiments.</p

HuPKR F489S confers resistance to VacV K3L H47R.

<p>HeLa PKR KO cells inducibly expressing the indicated PKR variants were infected and evaluated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.g003" target="_blank">Fig 3A</a>. (mean ± s.d., n = 3, ** p<0.005; ns, not significant). Data are representative of three independent experiments. (B) K3L was detected in lysates of the uninduced, infected empty vector cells from (A) by western blotting.</p

Position 489 of HuPKR is highly tolerant of amino acid substitutions.

<p>(A) Position 489 (dark blue) falls within the αG helix of PKR (light blue) and projects into a hydrophobic pocket of eIF2α (pale orange) composed of the side chains of Y32, E42, M44 and Y81 (dark orange). (B) Position 489 is highly variable in primates. The protein sequence alignment of the αG helix of PKR from representative primates is shown. Amino acids previously found to be evolving under positive selection among primates [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.ref016" target="_blank">16</a>] are indicated with arrowheads. (C) Most mutations at position 489 retain PKR activity but only a subset are inhibited by HCMV_TRS1. HuPKR 489 variants were evaluated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.g002" target="_blank">Fig 2B</a> (mean ± s.d., n = 2). The fold change relative to a vector control is indicated for amino acids that are most sensitive to inhibition by HCMV_TRS1 (greater than 3 fold increase). KD PKR is a mutant of PKR (K296R) that lacks kinase activity. Data are representative of at least two independent experiments.</p

Species-specific differences in primate CMV TRS1 PKR antagonism.

<p>(A) HeLa PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV_TRS1, AgmCMV_TRS1, RhCMV_TRS1, or SmCMV_TRS1. At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.</p