Autophagy is a multistep process in which cytoplasmic components, including invading pathogens, are captured by autophagosomes that subsequently fuse with degradative lysosomes. Negative-strand RNA viruses, including paramyxoviruses, have been shown to alter autophagy, but the molecular mechanisms remain largely unknown. We demonstrate that human parainfluenza virus type 3 (HPIV3) induces incomplete autophagy by blocking autophagosome-lysosome fusion, resulting in increased virus production. The viral phosphoprotein (P) is necessary and sufficient to inhibition autophagosome degradation. P binds to SNAP29 and inhibits its interaction with syntaxin17, thereby preventing these two host SNARE proteins from mediating autophagosome-lysome fusion. Incomplete autophagy and resultant autophagosome accumulation increase extracellular viral production but do not affect viral protein synthesis. These findings highlight how viruses can block autophagosome degradation by disrupting the function of SNARE proteins.

Mitophagy is a form of autophagy that selectively removes damaged mitochondria. Impaired mitochondria can be tagged by the kinase PINK1, which triggers recruitment of the E3-ubiquitin ligase Parkin and subsequent mitochondrial sequestration within autophagosomes. We previously found that human parainfluenza virus type 3 (HPIV3) infection induces autophagy, but the type and mechanisms of autophagy induction remain unknown. Here, we show that matrix protein (M) of HPIV3 translocates to mitochondria and interacts with Tu translation elongation factor mitochondrial (TUFM). M-mediated mitophagy does not require the Parkin-PINK1 pathway but rather an interaction between M and the LC3 protein that mediates autophagosome formation. These interactions with both TUFM and LC3 are required for the induction of mitophagy and lead to inhibition of the type I interferon response. These results reveal that a viral protein is sufficient to induce mitophagy by bridging autophagosomes and mitochondria.

. Furthermore, we found that N L478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N 0 -P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis. Human parainfluenza virus type 3 (HPIV3) is a cytoplasmic, enveloped virus with a nonsegmented negative-strand (NNS) RNA genome that is classified in the Paramyxoviridae family, in the order Mononegavirales. It can cause severe respiratory tract diseases such as bronchiolitis, pneumonia, and croup in infants and young children (1). However, currently no valid antiviral therapy or vaccine is available. Thus, further exploration of its replication mechanism will be helpful in the development of novel therapeutic approaches. The RNA genome of HPIV3 consists of 15,462 nucleotides and is encapsidated by the nucleoprotein (N; 68 kDa) to form a helical nucleocapsid containing N-RNA that has the characteristic herringbone-like structure also observed in other Paramyxoviridae members (2-6). This N-RNA complex serves as a template to interact with the RNA-dependent RNA polymerase (RdRp) complex consisting of a large protein (L; 255 kDa) and a phosphoprotein (P; 90 kDa) cofactor; interaction between N-RNA and RdRp forms an active ribonucleoprotein (RNP) complex that is necessary for transcription and replication (2, 7) to generate six monocistronic mRNAs and an antigenome intermediate. P mRNA encodes a basic protein, designated C, via the translation of a ϩ1 open reading frame of P mRNA, which is responsible for inhibiting viral RNA synthesis as well as counteracting the host interferon signaling pathway (8, 9). A synergic association between the L-P and N-RNA templates would therefore determine the ability of the RNA polymerase complex to transcribe or replicate.Pairs of paramyxoviruses, such as HPIV3 and Sendai virus and canine distemper virus and measles virus, share about 50% nucleotide identity, despite the low level of sequence similarity among known paramyxovirus N genes by sequence comparisons (10)(11)(12). N consists of two major domains that are chemically opposite in nature: a highly conserved N-terminal core (about 80% of the sequence), which forms a globular body, and a hypervariable Cterminal tail (about 20% of the sequence), which extends from the N-terminal body (13). The N terminus contains all of the necessary components for N self-assembly and RNA binding to form N-RNA complex (14-16). Structural assays of the N-RNA complex of some NNS RNA viruses revealed that the RNA is sequestered between the N-and C-terminal lobes of the N-RNA complex (17, 18). The C terminus is mainly responsible for the binding of the N-RNA complex to P (3,(19)(20)(21). Thus, the C terminus is required for the binding of the N-RNA template to the RNA polymerase complex for viral RNA synthesis (22,23). Studies of the nucleocapsid of Sendai virus showed that deletion of the C-terminal fragment abroga...

SMYD2 is a histone methyltransferase that has been reported to be an important epigenetic regulator. This study aims to investigate SMYD2 as a prognostic indicator of clear cell renal cell carcinoma (ccRCC) and explore its role in tumorigenesis and multi-drug resistance.Methods: Tumor specimens, clinicopathologic information, and prognostic outcomes of 186 ccRCC patients from three hospitals in China were collected for SMYD2 immunohistochemistry staining, Kaplan-Meier analysis, and Cox proportional hazards-regression analysis. MicroRNA (miRNA)-microarray profiling identified differentially expressed miRNAs in renal cancer cells subjected to SMYD2 knockdown or treatment with the SMYD2 inhibitor AZ505. The effects of SMYD2 and candidate SMYD2-mediated miRNAs on renal cancer cell proliferation, migration, clonogenicity, and tumorigenicity were determined via cell-function assays and murine xenograft experiments. The half-inhibitory concentrations (IC50) of five antineoplastic drugs (cisplatin, doxorubicin, fluorouracil, docetaxel, and sunitinib) in AZ505-treated and control cells were calculated, and the effects of SMYD2 inhibition on P-glycoprotein (P-gP) expression and multiple-drug resistance were verified.Results: SMYD2 was overexpressed and acted as an oncogene in ccRCC. High SMYD2 expression correlated with a high TNM stage (P = 0.007) and early tumor relapse (P = 0.032). SMYD2 independently predicted a worse overall survival (P = 0.022) and disease-free survival (P = 0.048). AZ505 inhibited the binding of SMYD2 to the miR-125b promoter region (based on chromatin immunoprecipitation assays) and suppressed ccRCC cell migration and invasion by inhibiting the SMYD2/miR-125b/DKK3 pathway. SMYD2 and miR-125b inhibition acted synergistically with anticancer drugs via P-gP suppression in vitro and in vivo.Conclusions: These findings suggested that SMYD2 plays an important role in ccRCC development and could be a potential biomarker for the treatment and prognosis of RCC.

Objectives We herein aimed to explore whether growth arrest‐specific 5 (GAS5) promotes M1 macrophage polarization in childhood pneumonia and to investigate the underlying mechanism. Methods Relative GAS5 and miR‐455‐5p expression and suppressor of cytokine signaling 3 (SOCS3) messenger RNA level were examined using quantitative reverse transcription polymerase chain reaction. Protein expression of SOCS3 and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway‐related proteins was detected using western blot analysis. Luciferase activity assay was performed to test whether miR‐455‐5p could bind to GAS5 or SOCS3. The macrophage phenotype was determined using flow cytometry analysis and enzyme‐linked immunosorbent assay. Results The macrophage polarization toward the M2 phenotype was observed in peripheral blood from pneumonia children. Furthermore, GAS5 and SOCS3 expression were upregulated but miR‐455‐5p downregulated in human monocyte‐derived macrophages from pneumonia children compared with the control group. Furthermore, GAS5 acted as a sponge for miR‐455‐5p to facilitate SOCS3 expression. Moreover, miR‐455‐5p mimic and SOCS3 knockdown significantly reversed the GAS5 overexpression‐mediated suppression of the JAK2/STAT3 signaling and promotion of M1 polarization. Conclusion GAS5 promotes M1 macrophage polarization by acting as a competing endogenous RNA of miR‐455‐5p to facilitate SOCS3 expression in childhood pneumonia.

Background : The programmed death 1 (PD1)/programmed death ligand 1 (PDL1) targeted therapies have gained positive outcomes in several tumors, but the evidence of the expression and prognosis value of PD1/PDL1 in high risk prostate cancer was rare. Methods : Immunohistochemical analysis of PDL1/PD1 expression by a validated antibody was performed in a retrospectively collected high risk prostate cancer cohort who received adjuvant hormonal therapy (AHT) after radical prostatectomy (RP). The association between PDL1/PD1 expression and prognosis was determined. Results : In total, 127 patients were enrolled. 49.6% patients were considered PDL1-high expression while the PD1-positive expression proportion was 24.4%. High PDL1 and negative PD1 expression were significantly associated with lower prostate specific antigen (PSA) density (p=0.010 and p=0.033, respectively). Compared with the PDL1-low expression patients, the PDL1-high expression patients had significantly shorter time to PSA nadir (TTN) (P=0.001) and biochemical recurrence (BCR) (P=0.004). In Kaplan-Meier analysis, the PDL1-high expression group (p<0.0001) and the PDL1-high/PD1-negative expression group (p<0.0001) showed markedly lower BCR-free survival in localized disease. Univariate cause-specific Cox proportional hazard regression model concluded total PSA (p=0.047), PDL1-high-expression (p<0.001), PDL1-high/PD1-negative expression (p<0.001) were significant risk factors of shorter progression time to BCR in localized disease. PDL1-high-expression was the independent predictor of time to BCR in multiple Cox regression of all patients (Hazard ratio [HR]: 3.901; 95% Confidence interval [CI]: 1.287-11.824; p=0.016). Conclusions : PDL1 expression is not only highly prevalent in high-risk prostate cancer, but is also an independent biomarker in the prognosis of high-risk prostate cancer received AHT after RP. PDL1/PD1 targeted therapy might be a potentially adjuvant treatment option for high-risk prostate cancer after RP.

The kidney function of patients with chronic kidney disease (CKD) is impaired irreversibly. Organ transplantation is the only treatment to restore kidney function in CKD patients. The assessment of new potential therapeutic procedures relies heavily on experimental animal models, but it is limited by its human predictive capacity. In addition, the frequently used two-dimensional in vitro human renal cell models cannot replicate all the features of the in vivo situation. In this study, we developed a three-dimensional (3D) in vitro human renal organoid model from whole kidney cells as a promising drug screening tool. At present, the renal tissue generated from human pluripotent stem cells (hPSCs) exhibits intrinsic tumorigenicity properties. Here we first developed a 3D renal organoid culture system that originated from adult differentiated cells without gene modification. Renal organoids composed of multiple cell types were created under optimal experimental conditions and evaluated for morphology, viability and erythropoietin production. As a novel screening tool for renal toxicity, 3D organoids were exposed to three widely used drugs: aspirin, penicillin G and cisplatin. The study results showed this 3D renal organoid model can be used as a drug screening tool, a new in vitro 3D human kidney model, and provide hope for potential regenerative therapies for CKD.

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the M L302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M L302A mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. IMPORTANCEHuman parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the M L302A mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.