Early retinal microvascular abnormalities in patients with chronic kidney disease.

OBJECTIVE: To evaluate early retinal microvascular abnormalities in patients with chronic kidney disease (CKD) via optical coherence tomography angiography. METHODS: A cross-sectional study. Two hundred patients with CKD stage ≧3 were enrolled in the CKD group, and 50 age-matched healthy subjects were enrolled in the control group. Main outcome measures were the differences in parafoveal vessel densities in the superficial vascular plexus (SVP) and deep vascular plexus (DVP) between the CKD and control groups. RESULTS: The mean ages were 62.7 ± 10.1 in the CKD group and 61.9 ± 9.7 (P = 0.622) in the control group. The CKD group had reduced parafoveal vessel densities in SVP (46.7 ± 4.3 vs 49.7 ± 2.9, P < 0.001) and DVP (50.1 ± 4.1 vs 52. 6 ± 2.9, P < 0.001) when compared to those of the control group. In multiple linear regression models, age, diabetes, estimated glomerular filtration rate, and use of anti-hypertensive drugs were factors associated with vessel density in SVP, whereas age, diabetes, and smoking were factors associated with vessel density in DVP. CONCLUSION: Patients with CKD had reduced vessel densities in parafoveal SVP and DVP, as compared to that of control subjects. Microvasculature in the different retinal layers may be affected by different systemic factors.

Exosome-delivered and Y RNA-derived small RNA suppresses influenza virus replication.

BACKGROUND: Multiple interplays between viral and host factors are involved in influenza virus replication and pathogenesis. Several small RNAs have recently emerged as important regulators of host response to viral infections. The aim of this study was to characterize the functional role of hsa-miR-1975, a Y5 RNA-derived small RNA, in defending influenza virus and delineate the mechanisms. METHODS: We performed high throughput sequencing of small RNAs in influenza virus-infected cells to identify up- or down- regulated small RNA species. The expression of the most abundant RNA species (hsa-miR-1975) was validated by stem-loop reverse transcription-polymerase chain reaction (RT-PCR). Antiviral effects of hsa-miR-1975 were confirmed by Western Blot, RT-PCR and plaque assay. In vitro perturbation of hsa-miR-1975 combined with exosomes isolation was used to elucidate the role and mechanism of hsa-miR-1975 in the context of antiviral immunity. RESULTS: Small RNA sequencing revealed that hsa-miR-1975 was the most up-regulated small RNA in influenza virus-infected cells. The amount of intracellular hsa-miR-1975 increased in the late stage of the influenza virus replication cycle. The increased hsa-miR-1975 was at least partially derived from degradation of Y5RNA as a result of cellular apoptosis. Unexpectedly, hsa-miR-1975 mimics inhibited influenza virus replication while hsa-miR-1975 sponges enhanced the virus replication. Moreover, hsa-miR-1975 was secreted in exosomes and taken up by the neighboring cells to induce interferon expression. CONCLUSIONS: Our findings unravel a critical role of Y-class small RNA in host's defense against influenza virus infection and reveal its antiviral mechanism through exosome delivery. This may provide a new candidate for targeting influenza virus.

SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.

UNLABELLED: Small ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular gene Ubc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. By in vivo and in vitro SUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication. IMPORTANCE: SUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a "double indemnity" of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.

MCPIP1 suppresses hepatitis C virus replication and negatively regulates virus-induced proinflammatory cytokine responses.

Human MCP-1-induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.

Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry.

Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually "hijack," or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses.

Modification of small hepatitis delta virus antigen by SUMO protein.

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.

Correlation between higher-order aberrations and visual acuity recovery (CoHORT) after spectacles treatment for pediatric refractive amblyopia: A pilot study using iDesign measurement.

PURPOSE: To determine the correlation between higher-order aberrations (HOAs) and best-corrected visual acuity (BCVA) recovery speed after spectacles treatment using iDesign measurements in refractive amblyopic children. METHODS: This is a prospective case series. Children aged from 3 to 7 years with refractive amblyopia (Landolt C equivalent < 0.8) were recruited. All participants were followed for at least 6 months after full correction of the refraction error by spectacles. The HOAs were measured using iDesign before and after cycloplegia at first visit and at 3-month intervals. Then correlation between BCVA recovery after treatment for 6 months and HOAs was determined. RESULTS: We analyzed 24 eyes of 12 children (mean age, 4.5 years). Baseline mean BCVA was logarithm of minimal angle of resolution (logMAR) 0.335 (Landolt C equivalent 0.46), which improved to logMAR 0.193 (Landolt C equivalent 0.64) after treatment with full-correction spectacles for 6 months. The amblyopic eye BCVA recovery was negatively correlated with tetrafoil with/without cycloplegia (P = 0.006 and 0.022, respectively) and trefoil with cycloplegia (P = 0.049). CONCLUSIONS: trefoil and tetrafoil measured with iDesign negatively correlates with the BCVA recovery speed of refractive amblyopic eyes after spectacles treatment in this pilot study. The current study results may aid in further investigation for diagnosis and treatment of refractory refractive and idiopathic amblyopia.

Retinal neurovascular changes in chronic kidney disease.

PURPOSE: To examine retinal neurovascular changes in patients with chronic kidney disease (CKD). METHODS: Case-control study. A total of 171 CKD cases and 40 controls were recruited (mean age 62.9 ± 10.3 versus 60.8 ± 9.2, p = 0.257). Retinal neural parameters, including parafoveal retinal thickness (PfRT), macular ganglion cell complex thickness (GCCt), global loss volume (GLV), focal loss volume (FLV) and peripapillary retinal nerve fibre layer thickness (RNFLt), were measured using optical coherence tomography (OCT). Microvascular parameters, including foveal avascular zone size, vessel density over the parafoveal superficial vascular plexus (SVP-VD), parafoveal deep vascular plexus (DVP-VD) and radial peripapillary capillary (RPC-VD), were measured using OCT angiography. RESULTS: Chronic kidney disease (CKD) patients showed reduced PfRT, GCCt and RNFLt and increased GLV and FLV compared with the controls (all p < 0.005). Among patients with CKD, estimated glomerular filtration rate was an independent factor associated with PfRT (coefficient 0.19, p = 0.015), GCCt (coefficient 0.10, p = 0.006), GLV (coefficient - 0.08, p = 0.001), FLV (coefficient - 0.02, p = 0.006) and RNFLt (coefficient 0.15, p = 0.002). Parafoveal retinal thickness (PfRT), GCCt, GLV, FLV and RNFLt were correlated with SVP-VD (all p < 0.001) but not with DVP-VD (all p > 0.1). CONCLUSIONS: Chronic kidney disease (CKD) patients demonstrated a significant reduction in macular thickness and changes in retinal neural parameters. These changes were associated with the severity of CKD and correlated with the microvascular rarefaction in the parafoveal SVP.

Transcription of subgenomic mRNA of hepatitis delta virus requires a modified hepatitis delta antigen that is distinct from antigenomic RNA synthesis.

Hepatitis delta virus (HDV) contains a viroid-like, 1.7-kb circular RNA genome, which replicates via a double-rolling-circle model. However, the exact mechanism involved in HDV genome RNA replication and subgenomic mRNA transcription is still unclear. Our previous studies have shown that the replications of genomic and antigenomic HDV RNA strands have different sensitivities to alpha-amanitin and are associated with different nuclear bodies, suggesting that these two strands are synthesized in different transcription machineries in the cells. In this study, we developed a unique quantitative reverse transcription-PCR (qRT-PCR) procedure for detection of various HDV RNA species from an RNA transfection system. Using this qRT-PCR procedure and a series of HDV mutants, we demonstrated that Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation of small hepatitis delta antigen (S-HDAg) are important for HDV mRNA transcription. In addition, these three S-HDAg modifications are dispensable for antigenomic RNA synthesis but are required for genomic RNA synthesis. Furthermore, the three RNA species had different sensitivities to acetylation and deacetylation inhibitors, showing that the metabolic requirements for the synthesis of HDV antigenomic RNA are different from those for the synthesis of genomic RNA and mRNA. In sum, our data support the hypothesis that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that of genomic RNA synthesis and mRNA transcription, even though the antigenomic RNA and the mRNA are made from the same RNA template. We propose that acetylation and deacetylation of HDAg may provide a molecular switch for the synthesis of the different HDV RNA species.

Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

Hepatitis delta virus RNA replication.

Hepatitis delta virus (HDV) is a distant relative of plant viroids in the animal world. Similar to plant viroids, HDV replicates its circular RNA genome using a double rolling-circle mechanism. Nevertheless, the production of hepatitis delta antigen (HDAg), which is indispensible for HDV replication, is a unique feature distinct from plant viroids, which do not encode any protein. Here the HDV RNA replication cycle is reviewed, with emphasis on the function of HDAg in modulating RNA replication and the nature of the enzyme involved.