Hierarchical self-recognition and response in CSC and non-CSC micro-niches for cancer therapy.

Cancer stem cells (CSCs) characterized by self-renewal, invasiveness, tumorigenicity and resistance to treatment are regarded as the thorniest issues in refractory tumors. We develop a targeted and hierarchical controlled release nano-therapeutic platform (SEED-NPs) that self-identifies and responds to CSC and non-CSC micro-niches of tumors. In non-CSC micro-niche, reactive oxygen species (ROS) trigger the burst release of the chemotherapeutic drug and photosensitizer to kill tumor cells and reduce tumor volume by combining chemotherapy and photodynamic therapy (PDT). In CSC micro-niche, the preferentially released differentiation drug induces CSC differentiation and transforms CSCs into chemotherapy-sensitive cells. SEED-NPs exhibit an extraordinary capacity for downregulating the stemness of CD44+/CD24- SP (side population) cell population both in vitro and in vivo, and reveal a 4-fold increase of tumor-targeted accumulation. Also, PDT-generated ROS promote the formation of tunneling nanotubes and facilitate the divergent network transport of drugs in deep tumors. Moreover, ROS in turn promotes CSC differentiation and drug release. This positive-feedback-loop strategy enhances the elimination of refractory CSCs. As a result, SEED-NPs achieve excellent therapeutic effects in both 4T1 SP tumor-bearing mice and regular 4T1 tumor-bearing mice without obvious toxicities and eradicate half of mice tumors. SEED-NPs integrate differentiation, chemotherapy and PDT, which proved feasible and valuable, indicating that active targeting and hierarchical release are necessary to enhance antitumor efficacy. These findings provide promising prospects for overcoming barriers in the treatment of CSCs.

Reinforced Immunogenic Endoplasmic Reticulum Stress and Oxidative Stress via an Orchestrated Nanophotoinducer to Boost Cancer Photoimmunotherapy.

Cancer progression and treatment-associated cellular stress impairs therapeutic outcome by inducing resistance. Endoplasmic reticulum (ER) stress is responsible for core events. Aberrant activation of stress sensors and their downstream components to disrupt homeostasis have emerged as vital regulators of tumor progression as well as response to cancer therapy. Here, an orchestrated nanophotoinducer (ERsNP) results in specific tumor ER-homing, induces hyperthermia and mounting oxidative stress associated reactive oxygen species (ROS), and provokes intense and lethal ER stress upon near-infrared laser irradiation. The strengthened "dying" of ER stress and ROS subsequently induce apoptosis for both primary and abscopal B16F10 and GL261 tumors, and promote damage-associated molecular patterns to evoke stress-dependent immunogenic cell death effects and release "self-antigens". Thus, there is a cascade to activate maturation of dendritic cells, reprogram myeloid-derived suppressor cells to manipulate immunosuppression, and recruit cytotoxic T lymphocytes and effective antitumor response. The long-term protection against tumor recurrence is realized through cascaded combinatorial preoperative and postoperative photoimmunotherapy including the chemokine (C-C motif) receptor 2 antagonist, ERsNP upon laser irradiation, and an immune checkpoint inhibitor. The results highlight great promise of the orchestrated nanophotoinducer to exert potent immunogenic cell stress and death by reinforcing ER stress and oxidative stress to boost cancer photoimmunotherapy.

A neutrophil-mediated carrier regulates tumor stemness by inhibiting autophagy to prevent postoperative triple-negative breast cancer recurrence and metastasis.

Recurrence and metastasis after resection are still the main challenges in clinical treatment of breast cancer. Residual tumor and cancer stem-like cells are the primary culprits of recurrence and metastasis. Recent research studies indicate that autophagy is a cytoprotective mechanism of tumors, which maintains the stemness of cancer cells and promotes tumor proliferation and metastasis. Here, we constructed a "Trojan horse" using neutrophils as the carrier (PH-RL@NEs) to prevent the recurrence and metastasis of postoperative breast cancer. Neutrophils, as a "Trojan horse," can quickly respond to postoperative inflammation and accurately deliver drugs to the residual tumor site. The inflammation-triggered "Trojan horse" was then opened to release the liposomes containing the chemotherapeutic drug paclitaxel (PTX) and the autophagy inhibitor hydroxychloroquine (HCQ). We found that HCQ could effectively inhibit tumor cell autophagy, interfere with tumor epithelial-mesenchymal transition, and reduce the tumor stem cell-like population. In the orthotopic 4T1 postoperative recurrence models, PTX and HCQ synergistically killed tumors and regulated the stemness of tumor cells, thereby significantly inhibiting tumor recurrence and metastasis. Our work proved that the inhibition of autophagy to reduce tumor stemness is feasible and effective, which opens up a new prospect for postoperative tumor treatment. STATEMENT OF SIGNIFICANCE: The present study aimed to solve the issues of postoperative recurrence and metastasis of breast cancer and low efficiency of drug administration after surgery. For this purpose, we constructed neutrophils containing hydroxychloroquine (HCQ) and paclitaxel (PTX) co-loaded liposomes (PH-RL@NEs), which for the first time regulated the stemness of tumor cells by inhibiting autophagy, thereby inhibiting postoperative recurrence and metastasis of breast cancer cells. The results showed that PH-RL@NEs enhanced the targeted drug delivery efficiency, with the help of postoperative inflammation chemotaxis of neutrophils. HCQ effectively inhibited autophagy of tumor cells and reduced tumor stem cell-like cells, thus improving the therapeutic effect in the 4T1 in situ postoperative recurrence model.

A dual receptors-targeting and size-switchable "cluster bomb" co-loading chemotherapeutic and transient receptor potential ankyrin 1 (TRPA-1) inhibitor for treatment of triple negative breast cancer.

Oxidative-stress defense system stands for the vulnerability of tumor cells because of the stronger oxidative stress existing in tumor sites. TRPA-1 has been found to be overexpressed in various tumors, related to the tumor proliferation and metastasis, and promote reactive oxygen species (ROS) and chemotherapy tolerance through Ca2+-dependent anti-apoptotic pathway in recent studies, which provides a new anti-tumor approach to target oxidative-stress defense system. However, there are few studies on the mechanisms of TRPA-1 inhibition increasing the effectiveness of chemotherapy and inhibiting tumor metastasis. Here, in order to deliver drugs to the deep tumor where is full of stronger oxidative stress, a dual receptors-targeting and size-switchable "cluster bomb" co-loading doxorubicine (DOX) and TRPA-1 inhibitor AP-18 (DA-tMN) was designed. DSPE-PEG2000 micelles (M, ~10 nm) were connected to the master core of hyaluronic acid nanogels (N, ~100 nm) to realize HAase-responsive size-switchable and acquired targeting characteristics. Besides, tumor homing peptide tLyP-1 (t) was modified on the surface of micelles to further increase tumor accumulation. Our study showed that tLyP-1 modification enhanced tumor-targeting delivery of tLyP-1-modified micelles @ nanogels (tMN) in vitro and in vivo. Then, HAase responsive nanogel core realized the deep penetration of tMN in 4 T1 3D tumor spheres models and 4 T1 tumor-bearing mice models. In vitro anti-tumor and anti-metastasis mechanism studies indicated that AP-18 increased the sensitivity of tumor cells to DOX by inhibiting Ca2+ influx and AKT phosphorylation caused by DOX. Compared with DOX-loaded tLyP-1-modified micelles @ nanogels (D-tMN), DA-tMN had the enhanced anti-tumor and anti-metastasis effect in vitro and vivo. Furthermore, the further anti-metastasis mechanism studies showed that TRPA-1 inhibition downregulate the expression of N-cadherin and vimentin and upregulate the expression of E-cadherin, which suggested that metastases inhibition caused by TRPA-1 inhibition may be related to the inhibition of epithelial-mesenchymal transition (EMT) process.

Remodeling tumor immune microenvironment via targeted blockade of PI3K-γ and CSF-1/CSF-1R pathways in tumor associated macrophages for pancreatic cancer therapy.

Immunotherapy has exhibited great potential in cancer treatment. However, for immunosuppressive tumors such as pancreatic cancer, immunotherapy is far from satisfactory. PI3K-γ and colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) pathways are involved in the infiltration and polarization of immunosuppressive cells including M2 tumor associated macrophages (M2 TAMs), causing a suppressive tumor immune microenvironment (TIME) in pancreatic cancer. Herein, a M2 TAM targeting nanomicelle was developed to co-deliver PI3K-γ inhibitor NVP-BEZ 235 and CSF-1R-siRNA for specific TAMs reprogramming and antitumor immune responses activation. M2 TAM targeting peptide M2pep was modified on a mixed micelle, which was potent to co-encapsulate BEZ 235 and CSF-1R siRNA. The formulated nanomicelle increased M2 TAM targeting efficiency both in vitro and in vivo. Compared with single pathway blockade, dual blockade of PI3k-γ and CSF-1R demonstrated enhanced TAM remodeling effects by reducing M2 TAM level and elevating M1 TAM level, and also suppressed tumor infiltration of myeloid-derived suppressor cells (MDSCs). Consequently, the M2 TAM targeting reprogramming system activated antitumor immune responses and achieved enhanced anti-pancreatic tumor effects via PI3K-γ blockade and downregulation of CSF-1R. The M2pep modified nanomicelle provides a promising method for co-delivery of siRNA and small molecule inhibitor to M2 TAM. Dual inhibition of both PI3K-γ and CSF-1R can remodel TIME and activate antitumor immune responses synergistically, providing an alternative approach for pancreatic cancer treatment.

Enhanced anti-tumor and anti-metastasis therapy for triple negative breast cancer by CD44 receptor-targeted hybrid self-delivery micelles.

Tumor growth and metastasis are multistep processes regulated by multiple signaling pathways. The successful treatment of cancer largely depends on the ability to inhibit metastatic process. Multiphase inhibition of metastasis is a promising approach. Here, we described a targeting delivery system which was constructed by mixing hyaluronic acid-d-α-tocopheryl succinate (HA-TOS, HT) and low molecular weight heparin-TOS (LMWH-TOS, LT) to form a stable hybrid micelle (HT-LT), encapsulating chemotherapeutic drug doxorubicin (DOX). The prepared HT-LT NPs was about 125 nm in diameter with high drug encapsulation rate and continuous drug release behavior. We confirmed that HT-LT NPs exhibited an effective targeting ability both in vitro and in vivo using a 4T1 model that was attributed to HA binding to CD44 receptors. In addition, HT-LT NPs acted on different phases of the invasion-metastasis cascade and inhibited tumor cell migration and invasion, thus inhibiting tumor metastasis. This combinatorial strategy provided an alternative approach for triple negative breast cancer therapy.

Sequential depletion of myeloid-derived suppressor cells and tumor cells with a dual-pH-sensitive conjugated micelle system for cancer chemoimmunotherapy.

Myeloid-derived suppressor cells(MDSCs)are one of the most important immunosuppressive cells in tumor microenvironment, which also promote the development and progression of tumor cells. Nevertheless, due to the different distribution features of MDSCs and tumor cells, selective elimination of MDSCs and tumor cells in tumor microenvironment remain a great challenge. Here we have designed a dual-pH-sensitivity conjugated micelle system (PAH/RGX-104@PDM/PTX) that could deliver liver-X nuclear receptor (LXR) agonist RGX-104 and paclitaxel (PTX) to the perivascular region and tumor cells, respectively. Upon arrival at the acidic tumor microenvironment, the PAH/RGX-104@PDM/PTX undergo structure disintegration and capacitate coinstantaneous release of RGX-104 in the perivascular regions, leaving the intact PTX containing micelles PDM/PTX for tumor deep penetration. The released RGX-104 can be preferentially taken up by leukocytes, endothelial cells and macrophages which are nicely enriched in perivascular regions to active the LXR, and further reduces immunosuppressive MDSC levels. The remained small micelles carrying PTX enable deep tumor penetration as well as rapid specific drug release in the endosomal/lysosomal to kill tumor cells. PAH/RGX-104@PDM/PTX exhibits superior tumor accumulation as well as tumor penetration, and suppresses 74.88% in vivo tumor growth. More importantly, PAH/RGX-104@PDM/PTX has significantly alleviated tumor immunosuppression by eliminating MDSCs and increasing cytotoxic T lymphocytes infiltration. Our studies suggest that the dual-pH-sensitive codelivery nanocarrier not only cause apoptosis of cancer cells but also regulate the tumor immune environment to ultimately enhance the antitumor effect of CTLs through MDSCs depletion.

A size-shrinkable nanoparticle-based combined anti-tumor and anti-inflammatory strategy for enhanced cancer therapy.

Cancer-related inflammation can promote tumorigenesis, tumor growth and tumor metastasis in many types of cancers. Therefore, inhibiting cancer-related inflammation significantly improves cancer therapy. It has been reported that metformin (MET) inhibits the nuclear translocation of nuclear factor-κB (NF-κB), a key factor in cancer-related inflammation. However, the short half-life and the lack of tumor targeting limit the anti-inflammatory efficacy of MET in vivo. Herein, using pH-sensitive imine bonds, MET and the chemotherapy drug doxorubicin (DOX) were loaded onto size-shrinkable RGD-DGL-GNP nanoparticles (RDG NPs) for combination therapy. The RGD-MET-DGL-GNP nanoparticles (RMDG NPs) penetrated deep into the tumor to deliver MET and inhibit the NF-κB activity in tumor cells, which further decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expressions in tumor tissues and suppressed tumor cell proliferation. As a result, the co-administration of RGD-DOX-DGL-GNP (RDDG NPs) and RMDG NPs induced an improved therapeutic effect in a xenograft tumor model and a lipopolysaccharide (LPS)-induced pulmonary metastasis model with murine 4T1 breast cancer and CT26 colon cancer cells. Combining RDDG and RMDG NPs to simultaneously target tumors and cancer-related inflammation is a very effective anti-cancer strategy.

Postoperative sleep-disordered breathing in patients without preoperative sleep apnea.

BACKGROUND: Recently published data show that postoperative apnea-hypopnea index (AHI) is significantly increased in some patients without preoperative sleep apnea. These patients may be at risk of developing perioperative adverse events related to sleep-disordered breathing (SDB). The objective of this study was to investigate the incidence and predictors of postoperative moderate-to-severe SDB (AHI > 15 events/h) in patients without sleep apnea preoperatively. METHODS: In a prospective observational fashion, patients were invited to undergo sleep studies with a portable device (Embletta X100) preoperatively at home and postoperatively on the first and third night after surgery in the hospital or at home. The primary outcome was the incidence of postoperative moderate-to-severe SDB (AHI > 15 events/h) in non-sleep apnea patients (preoperative AHI ≤ 5 events/h). Logistic regression was used to evaluate the association of clinical factors and preoperative sleep parameters with the occurrence of postoperative moderate-to-severe SDB. RESULTS: A total of 120 non-sleep apnea patients completed the study, of which 31 (25.8% [95% confidence interval: 18.3%-34.6%]) patients were found to have AHI > 15 events/h on postoperative night 1 and/or postoperative night 3 (postoperative SDB group), and 89 (74%) patients had an AHI ≤ 15 events/h on both postoperative night 1 and 3 (postoperative non-SDB group). The patients in the postoperative SDB group were older (60 ± 13 vs 53 ± 12 years, P = 0.008) with more smokers (32.3% vs 15.7%, P = 0.048) and had a greater increase in the obstructive apnea index (adjusted P = 0.0003), central apnea index (adjusted P = 0.0012), and hypopnea index (adjusted P = 0.0004). Multivariate logistic regression analysis found that age and preoperative respiratory disturbance index (RDI) were significantly associated with the occurrence of postoperative moderate-to-severe SDB, P = 0.018 and P = 0.006, respectively. The sensitivity privilege cutoff of RDI at 4.9 events/h identified 70.2% to 96.4%patients developing postoperative moderate-to-severe SDB. CONCLUSIONS: At least 18.3% of non-sleep apnea patients developed moderate-to-severe SDB after surgery. Age and preoperative RDI were associated with the occurrence of postoperative moderate-to-severe SDB.

Alternative scoring models of STOP-bang questionnaire improve specificity to detect undiagnosed obstructive sleep apnea.

BACKGROUND: Obstructive sleep apnea (OSA) is common among surgical patients. The STOP-Bang questionnaire is a validated screening tool with a high sensitivity. However, its moderate specificity may yield fairly high false positive rate. We hypothesized that the specific combinations of predicting factors in the STOP-Bang questionnaire would improve its specificity. METHODS: After research ethics approval, consented patients were asked to complete the STOP-Bang questionnaire and then underwent sleep studies. The predictive performance of the STOP-Bang alternative scoring models was evaluated. Five hundred sixteen patients with complete data on the STOP-Bang questionnaire and polysomnography were reported. RESULTS: When the STOP-Bang score was ≥ 3 (any 3 positive items), the sensitivity and specificity for identifying moderate-severe OSA was 87% and 31%, respectively. The specificity for any 2 positive items from the 4 STOP questions plus BMI > 35 kg/m(2), male gender, or neck circumference > 40 cm for identifying moderate-severe OSA was 85%, 77%, and 79%, respectively. Compared with STOP-Bang score ≥ 3, the predicted probability for severe OSA of the specific combinations of STOP score ≥ 2 + male and STOP score ≥ 2 + BMI increased by 36% and 42%, respectively. For severe OSA, the specific combination of STOP score ≥ 2 + BMI + male demonstrated a specificity of 97% and 86% increase in predicted probability versus any 4 positive items of STOP-Bang questionnaire. CONCLUSIONS: The specific constellations of predictive factors improved the specificity of STOP-Bang questionnaire. For patients with STOP score ≥ 2, male gender, and BMI > 35 kg/m(2) were more predictive than age ≥ 50 and neck circumference > 40 cm.

Predictive performance of the STOP-Bang score for identifying obstructive sleep apnea in obese patients.

BACKGROUND: The loud Snoring, Tiredness, Observed apnea, high blood Pressure (STOP)-Body mass index (BMI), Age, Neck circumference, and gender (Bang) questionnaire is a validated screening tool for identifying obstructive sleep apnea in surgical patients. However, the predictive performance of the STOP-Bang score in obese and morbidly obese patients remains unknown. METHODS: Preoperative patients were approached for consent and were screened for obstructive sleep apnea (OSA) by the STOP questionnaire. Information concerning Bang was collected. Laboratory or portable polysomnography were performed in 667 patients. Patients with BMI of ≥ 30 kg/m(2) were defined as obese patients and ≥ 35 kg/m(2) as morbidly obese. The predictive parameters (sensitivity, specificity, and positive and negative predictive values) for the STOP-Bang score in obese and morbidly obese patients were analyzed. RESULTS: In 310 obese patients, a STOP-Bang score of 3 has high sensitivity of 90 % and high positive predictive value of 85 % for identifying obese patient with OSA. A STOP-Bang score of 4 had high sensitivity (87.5 %) and high negative predictive value (90.5 %) for identifying severe OSA, whereas a STOP-Bang score of 6 had high specificity (85.2 %) to identify severe OSA. The diagnostic odds ratio of a STOP-Bang score of 4 was 4.9 for identifying severe OSA. In 140 morbidly obese patients, a STOP-Bang score of 4 had high sensitivity (89.5 %) for identifying severe OSA. CONCLUSIONS: The STOP-Bang score was validated in the obese and morbidly obese surgical patients. For identifying severe OSA, a STOP-Bang score of 4 has high sensitivity of 88 %. For confirming severe OSA, a score of 6 is more specific.

Serum bicarbonate level improves specificity of STOP-Bang screening for obstructive sleep apnea.

BACKGROUND: The STOP-Bang questionnaire is a validated screening tool for the identification of surgical patients with obstructive sleep apnea (OSA). A STOP-Bang score ≥ 3 is highly sensitive but only moderately specific. Apnea/hypopnea during sleep can lead to intermittent hypercapnia and may result in serum bicarbonate (HCO3⁻) retention. The addition of serum HCO3⁻ level to the STOP-Bang questionnaire may improve its specificity. METHODS: Four thousand seventy-seven preoperative patients were approached for consent and screened by the STOP-Bang questionnaire. Polysomnography was performed and preoperative HCO3⁻ level was collected in 384 patients. Study participants were randomly assigned to a derivation or validation cohort. Predictive parameters (sensitivity, specificity, positive and negative predictive values) for STOP-Bang score and serum HCO3⁻ level were calculated. RESULTS: In the derivation cohort, with a STOP-Bang score ≥ 3, the specificity for all OSA, moderate/severe OSA, and severe OSA was 37.0%, 30.4%, and 27.7%, respectively. HCO3⁻ level of 28 mmol/L was selected as a cutoff for analysis. With the addition of HCO3⁻ level ≥ 28 mmol/L to the STOP-Bang score ≥ 3, the specificity for all OSA, moderate/severe OSA, and severe OSA improved to 85.2%, 81.7%, and 79.7%, respectively. Similar improvement was observed in the validation cohort. CONCLUSION: Serum HCO3⁻ level increases the specificity of STOP-Bang screening in predicting moderate/severe OSA. We propose a two-step screening process. The first step uses a STOP-Bang score to screen patients, and the second step uses serum HCO3⁻ level in those with a STOP-Bang score ≥ 3 for increased specificity.

A perioperative smoking cessation intervention with varenicline: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: The efficacy of perioperative tobacco interventions on long-term abstinence and the safety of smoking cessation less than 4 weeks before surgery is unclear. Our objective was to determine the efficacy and safety of a perioperative smoking cessation intervention with varenicline to reduce smoking in elective surgical patients. METHODS: In a prospective, multicenter, double-blind, placebo-controlled trial, 286 patients were randomized to receive varenicline or placebo. Both groups received in-hospital and telephone counseling during 12 months. The primary outcome was the 7-day point prevalence abstinence rate 12 months after surgery. Secondary outcomes included abstinence at 3 and 6 months after surgery. Multivariable logistic regression was used to identify independent variables related to abstinence. RESULTS: The 7-day point prevalence abstinence at 12 months for varenicline versus placebo was 36.4% versus 25.2% (relative risk: 1.45; 95%: CI: 1.01-2.07; P = 0.04). At 3 and 6 months, the 7-day point prevalence abstinence was 43.7% versus 31.9% (relative risk: 1.37; 95% CI: 1.01 to 1.86; P = 0.04), and 35.8% versus 25.9% (relative risk: 1.43; 95%: CI 1.01-2.04; P = 0.04) for varenicline versus placebo, respectively. Treatment with varenicline (odds ratio: 1.76; 95% CI: 1.03-3.01; P = 0.04), and preoperative nicotine dependence (odds ratio: 0.82, 95% CI: 0.68 to 0.98; P = 0.03) predicted abstinence at 12 months. The adverse events profile in both groups was similar except for nausea, which occurred more frequently for varenicline versus placebo (13.3% vs. 3.7%, P = 0.004). CONCLUSIONS: A perioperative smoking cessation intervention with varenicline increased abstinence from smoking 3, 6, and 12 months after elective noncardiac surgery with no increase in serious adverse events.

[Molecular basis of one-way serological reaction between SINV and XJ-160 virus].

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.

Interaction of E2 glycoprotein with heparan sulfate is crucial for cellular infection of Sindbis virus.

Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357-7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext(-/-) cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145-150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145-150 aa, in SINV cellular infection through the interaction with HS.

Substitutions of 169Lys and 173Thr in nonstructural protein 1 influence the infectivity and pathogenicity of XJ-160 virus.

An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.

Inability of human immunodeficiency virus type 1 produced in murine cells to selectively incorporate primer formula.

Attempts to use the mouse as a model system for studying AIDS are stymied by the multiple blocks to human immunodeficiency virus type 1 (HIV-1) replication that exist in mouse cells at the levels of viral entry, transcription, and Gag assembly and processing. In this report, we describe an additional block in the selective packaging of tRNA(3Lys) into HIV-1 produced in murine cells. HIV-1 and murine leukemia virus (MuLV) use tRNA(3Lys) and tRNA(Pro), respectively, as primers for reverse transcription. Selective packaging of tRNA(3Lys) into HIV-1 produced in human cells is much stronger than that for tRNA(Pro) incorporation into MuLV produced in murine cells, and different packaging mechanisms are used. Thus, both lysyl-tRNA synthetase and GagPol are required for tRNA(3Lys) packaging into HIV-1, but neither prolyl-tRNA synthetase nor GagPol is required for tRNA(Pro) packaging into MuLV. In this report, we show that when HIV-1 is produced in murine cells, the virus switches from an HIV-1-like incorporation of tRNA(3Lys) to an MuLV-like packaging of tRNA(Pro). The primer binding site in viral RNA remains complementary to tRNA(3Lys), resulting in a significant decrease in reverse transcription and infectivity. Reduction in tRNA(3Lys) incorporation occurs even though both murine lysyl-tRNA synthetase and HIV-1 GagPol are packaged into the HIV-1 produced in murine cells. Nevertheless, the murine cell is able to support the select incorporation of tRNA(3Lys) into another retrovirus that uses tRNA(3Lys) as a primer, the mouse mammary tumor virus.

The interaction of APOBEC3G with human immunodeficiency virus type 1 nucleocapsid inhibits tRNA3Lys annealing to viral RNA.

Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA(3)(Lys), the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA(3)(Lys) priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA(3)(Lys) to synthetic viral RNA in the absence or presence of hA3G. The effect of hA3G on the annealing of tRNA(3)(Lys) to viral RNA and the ability of tRNA(3)(Lys) to initiate reverse transcription was measured. Our results show the following. (i) Electrophoretic band shift and primer binding site assays show that hA3G reduces the annealing of tRNA(3)(Lys) 44 and 60%, respectively, but does not disrupt the annealed complex once formed. (ii) hA3G inhibits tRNA(3)(Lys) priming 70 to 80%. (iii) Inhibition of tRNA(3)(Lys) priming by hA3G requires an interaction between hA3G and NCp7 during annealing. Thus, annealing of tRNA(3)(Lys) is insensitive to hA3G inhibition when facilitated by a zinc finger mutant of NCp7 unable to interact with hA3G. NCp7-independent annealing of DNA to viral RNA also is insensitive to hA3G inhibition. These results indicate that hA3G does not sterically block tRNA(3)(Lys) annealing by binding to viral RNA. Annealing and priming are not affected by another RNA binding protein, QKI-6.

Inhibition of initiation of reverse transcription in HIV-1 by human APOBEC3F.

Vif-negative HIV-1 produced in non-permissive human cells incorporate both APOBEC3F (hA3F) AND APOBEC3G (hA3G), and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that this reduction is due to deamination of deoxycytidine in viral DNA by either hA3G or hA3F, followed by DNA degradation, recent evidence indicates that the inhibition of viral DNA production can occur independently of DNA editing by either hA3F or hA3G. We have reported that the presence of hA3G in Vif-negative HIV-1 produced from either the non-permissive cell line, H9, or from transfected 293T cells transiently or stably expressing hA3G, results in a >or=50% reduction in the ability of primer tRNA(Lys3) to initiate reverse transcription in these virions, and that this is correlated with a similar reduction in the production of early DNA transcripts in the infected cells. In this work, we show that, like hA3G, hA3F in Vif-negative virions also results in a similar reduction in the initiation of reverse transcription in HIV-1, which is correlated with the inhibition of early viral DNA synthesis in the cell, and which does not require cytidine-deamination of DNA.

[Molecular characteristics of three new isolates of Japanese encephalitis virus in Fujian Province].

OBJECTIVE: To analyze the genotype of newly isolated Japanese encephalitis viruses in Fujian province and the characteristics of amino acid sequence in the E gene. METHODS: PrM and E segments of newly isolated Japanese encephalitis viruses were amplified by RT-PCR, the PCR products were cloned into T vector for sequencing. Phylogenetic analysis was carried out by PHYLIP program. RESULTS: Newly isolated Japanese encephalitis viruses belonged to genotype III, the nucleotide and amino acid of E gene showed high homology to vaccine strain SA14-14-2, but had some difference in some domains. CONCLUSION: Newly isolated viruses in Fujian province belonged to Japanese encephalitis virus genotype III. E protein of the isolates showed some differences as compared with vaccine strains.