Japanese Encephalitis Virus Core Protein Inhibits Stress Granule Formation through an Interaction with Caprin-1 and Facilitates Viral Propagation

Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys⁹⁷ and Arg⁹⁸ in the α-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys⁹⁷ and Arg⁹⁸ were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.     

APOBEC3G Oligomerization Is Associated with the Inhibition of Both Alu and LINE-1 Retrotransposition

Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A–H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.     

Deep Sequencing Reveals Low Incidence of Endogenous LINE-1 Retrotransposition in Human Induced Pluripotent Stem Cells

Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3′ end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.     

应激颗粒的形成与生物学意义

真核细胞对外界压力刺激会做出一系列应答反应,如暂停蛋白质翻译系统,从而使细胞能更好地适应环境压力。通过应激颗粒（stress granules,SG）的形成包裹未被翻译的mRNA是该适应性调节的重要方式。研究表明,环境压力导致eIF2α上游激酶的激活从而磷酸化eIF2α,翻译起始受阻,随后,TIA-1、TTP等蛋白迅速与mRNP结合聚集成SG,并在微管蛋白的帮助下进一步向细胞核聚集,形成成熟的SG。当压力消失,SG依赖微管及其动力蛋白进行解聚,释放包裹的mRNA及蛋白。细胞内成熟的SG在转录后调节中发挥重要作用,并且通过其组成蛋白在肿瘤凋亡、病毒侵染、免疫、炎症反应及由蛋白错误折叠引起的疾病中发挥作用。该文首次综述了压力颗粒研究进展,为充分认识SG的病理生理性调节功能提供参考。     

DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).     

Ubiquitin-Associated (UBA) Domain in Human Fas Associated Factor 1 Inhibits Tumor Formation by Promoting Hsp70 Degradation

Human Fas associated factor 1 (hFAF1) is a pro-apoptotic scaffolding protein containing ubiquitin-associating (UBA), ubiquitin like 1 and 2 (UBL1, UBL2), and ubiquitin regulatory X (UBX) domains. hFAF1 interacts with polyubiquitinated proteins via its N-terminal UBA domain and with valosin containing protein (VCP) via its C-terminal UBX domain. Overexpression of hFAF1 or its N-terminal UBA domain significantly increases cell death by increasing the degradation of polyubiquitinated proteins. In this study, we investigated whether hFAF1, whose expression level is reduced in cervical cancer, plays a role in tumor formation. We found that HeLa cells overexpressing full-length hFAF1 or the hFAF1 UBA domain alone, significantly suppressed the anchorage independent tumor growth in soft agar colony formation, increased cell death, and activated JNK and caspase 3. Employing UBA-specific tandem immunoprecipitation, we identified moieties specifically interacting with UBA domain of hFAF1, and found that polyubiquitinated Hsp70s are recruited to UBA domain. We also demonstrated that hFAF1 overexpression promotes Hsp70 degradation via the proteasome. We further found that mutating the UBA domain (I41N), as well as knocking down hFAF1 with specific RNAi, abolishs its ability to increase the proteasomal degradation of Hsp70. These findings suggest that hFAF1 inhibits tumor formation by increasing the degradation of Hsp70 mediated via its UBA domain.     

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3 end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.     

An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.     

The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M₁-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.