Functional cross-talk between distant domains of chikungunya virus non-structural protein 2 is decisive for its RNA-modulating activity.

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5'-3' directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.

Genetic ablation of arginase 1 in macrophages and neutrophils enhances clearance of an arthritogenic alphavirus.

Chikungunya virus (CHIKV) and Ross River virus (RRV) cause a debilitating, and often chronic, musculoskeletal inflammatory disease in humans. Macrophages constitute the major inflammatory infiltrates in musculoskeletal tissues during these infections. However, the precise macrophage effector functions that affect the pathogenesis of arthritogenic alphaviruses have not been defined. We hypothesized that the severe damage to musculoskeletal tissues observed in RRV- or CHIKV-infected mice would promote a wound-healing response characterized by M2-like macrophages. Indeed, we found that RRV- and CHIKV-induced musculoskeletal inflammatory lesions, and macrophages present in these lesions, have a unique gene-expression pattern characterized by high expression of arginase 1 and Ym1/Chi3l3 in the absence of FIZZ1/Relmα that is consistent with an M2-like activation phenotype. Strikingly, mice specifically deleted for arginase 1 in neutrophils and macrophages had dramatically reduced viral loads and improved pathology in musculoskeletal tissues at late times post-RRV infection. These findings indicate that arthritogenic alphavirus infection drives a unique myeloid cell activation program in inflamed musculoskeletal tissues that inhibits virus clearance and impedes disease resolution in an arginase 1-dependent manner.

Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak.

BACKGROUND: Sindbis viral vectors are able to efficiently target and kill tumor cells in vivo, as shown using pancreatic and ovarian cancer models. Infection results in apoptosis both in vitro and in vivo. Sindbis vector uptake is mediated by the LAMR, which is upregulated on a number of different tumor types, thus conferring specificity of the vector to a wide range of cancers. In this study we elucidate the mechanism of apoptosis in two tumor cell lines, MOSEC, derived from the ovarian epithelium and Pan02, derived from a pancreatic adenocarcinoma. A comprehensive understanding of the mechanism of apoptosis would facilitate the design of more effective vectors for cancer therapy. RESULTS: The initial phase of Sindbis vector induced apoptosis in MOSEC and Pan02 models reconfirms that viral infection is sensed by PKR due to double-stranded RNA intermediates associated with genomic replication. PKR activation results in translation inhibition through eIF2alpha phosphorylation and initiation of the stress response. Our studies indicate that the roles of two proteins, Mcl-1 and JNK, intimately link Sindbis induced translational arrest and cellular stress. Translational arrest inhibits the synthesis of anti-apoptotic Bcl-2 protein, Mcl-1. JNK activation triggers the release of Bad from 14-3-3, which ultimately results in apoptosis. These signals from translational arrest and cellular stress are propagated to the mitochondria where Bad and Bik bind to Bcl-xl and Mcl-1 respectively. Formation of these heterodimers displaces Bak, which results in caspase 9 cleavage and signaling through the mitochondrial pathway of apoptosis. CONCLUSION: The host cell response to Sindbis is triggered through PKR activation. Our studies demonstrate that PKR activation and subsequent translational arrest is linked to both cellular stress and apoptosis. We have also found the linkage point between translational arrest and apoptosis to be Mcl-1, a protein whose constant translation is required for inhibition of apoptosis. With this information vectors can be designed, which express or repress proteins implicated in this study, to enhance their therapeutic potential.

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells.

The alphavirus non-structural protein 3 (nsP3) has a conserved N-terminal macro domain and a variable highly phosphorylated C-terminal domain. nsP3 forms complexes with cellular proteins, but its role in virus replication is poorly understood and protein interaction domains have not been defined. As the N-terminal macro domain can bind poly(ADP-ribose) (PAR), and PAR polymerase-1 (PARP-1) is activated and autoribosylated during Sindbis virus (SINV) infection, it was hypothesized that PARP-1 and nsP3 may interact. Co-immunoprecipitation studies showed that PARP-1 interacted with nsP3 during SINV infection of NSC34 neuronal cells and was most abundantly present in replication complexes that contained plus- and minus-strand SINV RNAs 10-14 h after infection, prior to PARP-1 activation or automodification with PAR. Treatment with an inhibitor of PARP enzymic activity did not affect the interaction between nsP3 and PARP-1 or SINV replication. Co-expression of individual domains of nsP3 with PARP-1 showed that nsP3 interacted with PARP-1 through the C-terminal domain, not the N-terminal macro domain, and that phosphorylation was not required. It was concluded that PARP-1 interacts with the C-terminal domain of nsP3, is present in replication complexes during virus amplification and may play a role in regulating virus RNA synthesis in neuronal cells.

Inhibition of Chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the E2 surface glycoprotein.

Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus that causes in humans an acute infection characterized by polyarthralgia, fever, myalgia, and headache. Since 2005 this virus has been responsible for an epidemic outbreak of unprecedented magnitude. By analogy with other alphaviruses, it is thought that cellular proteases are able to process the viral precursor protein E3E2 to produce the receptor-binding E2 protein that associates as a heterodimer with E1. Destabilization of the heterodimer by exposure to low pH allows viral fusion and infection. We show that among a large panel of proprotein convertases, membranous furin but also PC5B can process E3E2 from African CHIKV strains at the HRQRR(64) / ST site, whereas a CHIKV strain of Asian origin is cleaved at RRQRR(64) / SI by membranous and soluble furin, PC5A, PC5B, and PACE4 but not by PC7 or SKI-1. Using fluorogenic model peptides and recombinant convertases, we observed that the Asian strain E3E2 model peptide is cleaved most efficiently by furin and PC5A. This cleavage was also observed in CHIKV-infected cells and could be blocked by furin inhibitor decanoyl-RVKR-chloromethyl ketone. This inhibitor was compared with chloroquine for its ability to inhibit CHIKV spreading in myoblast cell cultures, a cell-type previously described as a natural target of this virus. Our results demonstrate the role of furin-like proteases in the processing of CHIKV particles and point out new approaches to inhibit this infection.

Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses.

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Inhibition of matrix metalloproteinases ameliorates blood-brain barrier disruption and neuropathological lesions caused by avirulent Semliki Forest virus infection.

Semliki Forest virus (SFV) infection of mice is a useful model of viral neuropathogenesis in animals and avirulent strains such as SFV-A7 induce immune-mediated demyelination and death of neurones by necrosis and apoptosis. Matrix metalloproteinases (MMPs) have been implicated in various diseases including arthritis and cancer in many species. In this report, we show that MMP-2 and MMP-9 expression is induced in the brains of mice infected i.n. with SFV-A7. Treatment of mice with the pan MMP inhibitor GM6001 ameliorated the development of SFV-induced neuropathological lesions via an effect on the integrity of the blood-brain barrier. Low levels of neuronal necrosis and demyelination in GM6001-treated mice correlated with localisation of fibrinogen staining to thin-walled blood vessels and less intense staining of the perivascular neuropil.