[Prognostic value of skeletal muscle measured by CT at the T4 level in advanced EGFR-positive non-small cell lung cancer patients treated with ecotinib].

Objective: To investigate the prognostic value of skeletal muscle measured by CT at the level of the fourth thoracic vertebra (T4) in advanced epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (NSCLC) patients treated with ecotinib. Methods: The study retrospectively reviewed clinical and pathological characteristics of 176 patients with advanced EGFR-positive NSCLC who received ecotinib and underwent chest CT scans at Wuhan Union Hospital between January 2017 and October 2020. Among them, 70 were male and 106 were female, with ages ranging from 27 to 80 (58.6±10.6) years. As of August 21, 2021, the median follow-up duration was 19.2 months (95%CI: 15.3 to 23.7 months). The optimal cut-off value of skeletal muscle density (T4-SMD) on CT images at the T4 level were determined using X-tile software. Kaplan-Meier analysis and log-rank test were used to plot progression-free survival curves. Cox proportional hazards regression models were employed to analyze factors influencing 1-year progression-free survival (PFS), and a nomogram prognostic model was constructed accordingly. Receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA) were utilized to evaluate the predictive value of the nomogram. Results: The T4-SMD [M (Q1,Q3)] of 176 patients was 42.56 (37.05, 45.93) HU. Patients were divided into low T4-SMD group (n=122) and high T4-SMD group (n=54) based on the cut-off value (The values for males and females were 49.44 and 41.41 HU, respectively) of T4-SMD. The median PFS time and 1-year PFS rate in the low T4-SMD group were significantly lower than those in the high T4-SMD group [10.4 (95%CI: 9.3-11.8) vs 13.7 (95%CI: 11.1-18.5) months, 36.1% vs 59.3%, respectively, P=0.034]. Eastern Cooperative Oncology Group performance status (HR=3.308, 95%CI: 1.183-9.247, P=0.023), lactate dehydrogenase level (HR=1.852, 95%CI: 1.037-3.307, P=0.037), systemic immune-inflammation index (HR=1.772, 95%CI: 1.019-3.080, P=0.043), and T4-SMD (HR=0.563, 95%CI: 0.325-0.974, P=0.040) were prognostic factors for 1-year PFS in advanced EGFR-positive NSCLC patients treated with ecotinib. A nomogram for predicting 1-year PFS of advanced EGFR-positive NSCLC patients treated with ecotinib was constructed based on the four indicators selected by multivariate Cox regression analysis. The area under the ROC curve of the nomogram was 0.775 (95%CI: 0.676-0.874). The calibration curve showed good consistency between the predicted and actual 1-year PFS. DCA demonstrated good clinical prediction effectiveness of the nomogram. Conclusion: Low T4-SMD is a prognostic risk factor for patients with advanced EGFR-positive NSCLC receiving icotinib therapy.

Statins improve asthma symptoms by suppressing inflammation: a meta-analysis based on RCTs.

OBJECTIVE: Asthma is a chronic airway inflammatory disease caused by high infiltration of multiple inflammatory cells and factors in airway tissues. Statins may inhibit inflammation, hence improve asthma symptoms. This meta-analysis aimed to assess the efficacy of statins in the treatment of asthma patients. MATERIALS AND METHODS: We searched in PubMed, OVID, Cochrane Library, and Web of Science databases using the following key words: "statin", "atorvastatin", "simvastatin", "pravastatin", "rosuvastatin", "pitavastatin", "fluvastatin", and "asthma". The effects of statins on function, serum biomarkers, sputum mediators, and serum biochemical markers were analyzed from the identified studies. RESULTS: Twelve articles (613 participants) were included in the meta-analysis. Results showed that statins significantly improved asthma symptoms (ACQ score: MD: -0.43, 95% CI: -0.47 - -0.38, p<0.01; ACT score: MD: 1.96, 95% CI: 1.26-2.67, p<0.01). Furthermore, statins significantly reduced serum hsCRP (MD: -0.50, p=0.02) and cholesterol (MD: -32.76, p<0.01) levels and the proportion of sputum eosinophils (MD: -1.25, p<0.01) and IL6 levels (MD: -64.56, p=0.04) in sputum. However, lung function was not significantly different between the statin and placebo treatment groups. CONCLUSIONS: Although statins did not change the lung function in patients with asthma, they improved asthma symptoms and reduced the serum hsCRP, sputum eosinophil ratio, and IL6 levels.

[Comparison of the efficacy of PCSK9 inhibitors and statins on dyslipidemia in patients with type 2 diabetes].

Objective: To compare the efficacy of preprotein convertase subtilisin lysozyme 9 (PCSK9) inhibitors with statins in patients with type 2 diabetes mellitus (T2DM). Methods: A total of 140 patients with T2DM (80 males and 60 females) in the People's Hospital Affiliated to Shandong First Medical University from January 2018 to January 2021 were selected, with a mean age of (55±5) years (41-72 years). The patients were divided into observation group (n=68) and control group (n=72) by the random number table method. Both groups were given conventional treatments such as hypoglycemic drugs, the control group was given statins to regulate lipids, and the observation group was given PCSK9 inhibitors to lower lipids. The differences of low-density lipoprotein cholesterol (LDL-C), interleukin (IL)-1, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) expression levels and standard-reaching rate of LDL-C between the two groups were compared. The correlation between serum PCSK9 level and fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) and other indicators in T2DM patients was analyzed by Pearson correlation analysis. Results: After treatment, the LDL-C of the observation group was (2.3±0.7) mmol/L, which was lower than that of the control group [(2.7±0.7) mmol/L] (P=0.024); the standard-reaching rate of LDL-C of the observation group was 89.7% (61/68), which was higher than that of the control group [68.1% (49/72)] (P=0.002); the levels of IL-1, IL-6, TNF-, CRP, IL-10 and IL-8 in the observation group after treatment were (27.6±6.6) ng/L, (36.7±6.9) ng/L, (40.1±8.9) ng/L, (7.8±1.8) ng/L, (19.2±3.3) ng/L, (13.7±3.3) ng/L, respectively, which were lower than those in the control group [(30.6±7.9) ng/L, (40.1±7.3) ng/L, (43.4±9.2) ng/L, (10.4±2.5) ng/L, (30.7±3.7) ng/L, (26.8±3.4) ng/L, respectively] (all P<0.05). After treatment, the PCSK9 level in the observation group was (74±13) µg/L, which was lower than that in the control group [(97±14) µg/L] (P<0.001). The level of PCSK9 in T2DM patients was positively correlated with LDL-C, IL-1, IL-6 and TNF-α (r=0.390, 0.433, 0.398 and 0.562, all P<0.05). Conclusion: PCSK9 inhibitors have better lipid-regulating effects in patients with T2DM and can improve the level of inflammation at the same time.

[Clinical characteristics of patients with acute pulmonary embolism in high altitude area of Yunnan province in China].

Objective: To analyze the clinical features of patients with acute pulmonary embolism (APE) living in high altitude area of Yunnan province. Methods: This was a cross-sectional retrospective study. APE patients, hospitalized in our hospital between January 2017 and December 2019, were included. The selected patients were divided into low-risk group, medium-risk group and high-risk group according to risk stratification. The clinical data of patients, including demographic data, the main symptoms, risk factors of APE, heart rate and systolic blood pressure and laboratory testing results (D-dimer, cardiac troponin I (cTNI), N terminal B-type natriuretic peptide (NT-proBNP)) and echocardiography and electrocardiogram examination results, were obtained through the electronic medical record system. The clinical characteristics of selected patients were analyzed. Results: A total of 392 patients, aged (63.5±15.7) years, 224 males (57.14%), were included in this study and there were 59 low-risk, 304 medium-risk and 29 high-risk patients in this cohort. The main clinical manifestations were chest pain (157(40.05%)), dyspnea (107(27.30%)), hemoptysis (55(14.03%)), syncope as the first symptom (20(5.10%)), and only 6 cases (1.53%) presented with the typical "Virchow's triad". Most of the patients were accompanied by atypical chest tightness (223(56.89%)) and cough (208(53.06%)). The main risk factors were venous thrombosis of lower limbs (179(45.66%)), hypertension (138(35.20%)), surgery (63(16.07%)), and chronic obstructive pulmonary disease (COPD) (62(15.82%)). There were 57 cases (14.54%) of coronary heart disease, 57 cases (14.54%) of diabetes, 51 cases (13.01%) of cerebral infarction, 47 cases (12.00%) of advanced age, 15 cases (3.83%) of tumor, 7 cases (1.79%) of activity restriction, 6 cases (1.53%) of pregnancy and 4 cases (1.02%) of hormone use in this cohort. The proportion of lower extremity venous thrombosis was significantly higher in low-risk group than in medium-risk group (P<0.01), COPD was more common in high-risk and medium-risk groups than in low-risk group (P<0.01), hypertension was more common in high-risk group than in medium-and low-risk groups (P<0.01). The proportion of advanced age was significantly higher in medium-risk group than in low-risk group (P<0.01). There were no significant differences in RBC and hemoglobin level between low-, medium-and high-risk groups (P>0.05). The level of D-dimer was significantly higher in high-risk group than in medium-and low-risk groups (P<0.05). Levels of NT-proBNP and cTNI were significantly higher in high-risk group than in medium- and low-risk groups (P<0.05). Increased proportion of cTNI and NT-proBNP was significantly higher in high-risk group than in medium- and low-risk groups (P<0.05). There were 105 (26.79%) patients with pulmonary hypertension (PAH). The incidence of PAH was significantly higher in high-risk group than in low-risk group (P<0.01). There were 104 patients (26.53%) with right ventricular enlargement, and the incidence of right ventricular enlargement was significantly higher in high-risk group than in medium-and low-risk groups (P<0.01). Characteristic changes of electrocardiogram in patient with APE were T-wave inversion of limb leads (98(25.00%)), followed by SⅠQⅢTⅢ (83(21.17%)). Conclusions: The main clinical manifestations of APE in Yunnan high altitude area are chest pain and dyspnea, and syncope is the first symptom in some patients, but the typical "Virchow's triad" is rare. The most common risk factors are lower extremity venous thrombosis, hypertension, and COPD. Clinical symptoms, risk factors and laboratory examination results differ among patients with different risk stratification.

[Effects of occupational exposure to aluminum on verbal function of workers].

Objective: To explore the effect of occupational aluminum (Al) exposure on workers' overall cognitive level and speech function. Methods: In July 2019, by using cluster sampling method, the group of 232 exposed to Al from an Al plant in Shanxi Province were selected as the exposed group, and the group of 228 not exposed to Al were selected as the control group. The blood Al concentration was determined by inductively coupled plasma mass spectrometry (ICP-MAS) . The exposed group was divided into high exposure group and low exposure group according to the median (M) concentration of Al in serum. The general cognitive function and speech function were evaluated with the Montreal Cognitive Assessment Scale (MoCA) , and the differences in the general cognitive function and speech function between the groups were compared, and non-conditional logistic regression was used to analyze the possible influencing factors of mild cognitive impairment (MCI) . Results: There were significant differences in age, working age, education and drinking status between Al exposed group and control group (P<0.05) . The total MoCA score, animal naming tese (ANT) score and category fluency repetition (CFT) score in Al exposure group were lower than control group and the differences were statistically significant (P<0.05) . The full rate of ANT was lower than that of CFT in Al exposure group (P<0.05) . The total MoCA score, ANT score and CFT score in the high exposure group were significantly lower than those in the control group (P<0.05) . The score of MoCA, ANT and CFT in high exposure group were lower than those in low exposure group between the level of serum aluminum>60 µg/L group and ≤60 µg/L group (P<0.05) . Logistic regression analysis showed that working age, serun Al concentration, ANT score, CFT score and SR score were the possible influencing factors of MCI in occupational Al exposure workers (P<0.05) . Conclusion: Occupational Al exposure can lead to impaired speech function of workers, the higher the ANT score, CFT score and SR score, the lower working age and serum Al concentration, the lower risk of MCI.

[The impact of occupational aluminum exposure on workers' overall cognitive function and various cognitive fields].

Objective: To evaluate the effects of occupational aluminum exposure on workers' overall cognitive function and cognitive fields. Methods: From July to August 2019, using the method of cluster sampling, 101 and 117 workers were selected from the electrolytic aluminum workshop of an aluminum plant in a region and the maintenance workshop of a plant in the same region. The venous blood of the subjects was collected, the plasma was extracted, and the blood aluminum concentration was measured by ICP-MS. According to the blood aluminum concentration and type of work, 93 workers who were lower than the median blood aluminum concentration and in the maintenance workshop of a factory were divided into low aluminum exposure group, and 85 workers who were higher than the median blood aluminum concentration and in the electrolytic aluminum workshop of an aluminum factory were divided into high aluminum exposure group. The basic information of the respondents, was collected through the employee physical examination form. The overall cognitive function of workers was evaluated by Beijing Montreal Cognitive Assessment Scale (MoCA) . Multiple linear regression analysis and logistic regression analysis were used for multiple statistical analysis. Results: Compared with the low aluminum exposure group (25.42±1.808) , the total score of MoCA in the high aluminum exposure group (23.84±2.919) was significantly lower, and the scores of visual space, executive function, abstract and delayed recall were significantly lower (P<0.05) . Linear regression analysis showed that the total score of MoCA, visual space and executive function, naming and delayed recall were negatively correlated with blood aluminum concentration (ß=-0.018、-0.008、-0.003、-0.008, P<0.05) .MOCA total score, visual space and executive function, attention, language, abstraction, orientation were positively correlated with educational level (ß=0.853、0.310、0.216、0.171、0.412、0.122, P<0.05) . Logistic regression analysis showed that adjusting for age, smoking, drinking and education, blood aluminum was a risk factor for mild cognitive impairment (OR=1.017, P<0.05) ; Education level was the protective factor of mild cognitive impairment (OR=0.419, P<0.05) . Conclusion: Occupational aluminum exposure can affect the overall cognitive function of workers, and occupational aluminum exposure increases the risk of MCI.

[Clinical characteristics analysis of children with reversible posterior leukoen-cephalopathy syndrome during the treatment of hematological tumor].

OBJECTIVE: To analyze the clinical characteristics of patients with hematological tumor or disease before and after reversible posterior leukoen-cephalopathy syndrome (RPLS). METHODS: Five patients were both from Peking University First Hospital Pediatric Hematology-oncology Department in the period from March 2012 to March 2017. The gender, age, BMI, underlying diseases, with or without renal damage, hypertension family history, clinical manifestations of convulsions, hemoglobin, and blood pressure, serum sodium levels before and after convulsion, and other data of the children with RPLS were retrospectively analyzed. In the meantime, we followed up the five patients for 6 months to 66 months, kept a watchful eye on their original condition and the recovery of symptoms and signs of the nervous system. The relevant literature was reviewed. RESULTS: All of the subjects were females in school-age or pre-school age. The underlying diseases were malignant tumor associated with renal involvement or on one side of nephrectomy in 4 of these subjects, while the other one was refractory autoimmune hemolytic anemia. All of the subjects suffered from mild or moderate anemia. The day before RPLS occurred they received chemotherapy made up with cyclophosphamide, vincristine, and actinomycin-D, or the therapy with cyclosporin A and glucocorticoid. The clinical manifestations were afebrile convulsion after getting up in the mooring or in the afternoon. We observed elevation of blood pressure and cutting down of serum sodium compared with themselves. All of the cases recovered soon after management with diazepam, furosemide and amlodipine besylate. Four of them had a good outcomes and did not remain any sequela, while only one girl became childish in emotion and behavior, and then returned gradually to normal two years later. However, by long-term follow-up, the elevation of blood pressure was mainly reviewed in literature. CONCLUSION: The patients attacked by RPLS, with hematology or oncology cases, could have the underlying disease of renal damage and anemia. Blood pressure elevation and serum sodium falling down at the same time may play an important role during the occurrence of RPLS. Remaining stable of blood pressure and electrolyte level together will possibly reduce or mitigate RPLS.

[The effect of aluminum trichloride on expression of phosphorylated tau and Aß in SH-SY5Y cells].

Objective: To investigate the expression of phosphorylated tau (p-tau) and Aß in SH-SY5Y cells induced by aluminum or/and ApoE ε4 allele, and study the interaction between aluminum and ApoE ε4 allele. Methods: SH-SY5Y cells were assigned to control group, 400 µmol/L AlCl(3) group, ApoEε4 transfected group and 400 µmol/L AlCl(3) with Apo Eε4 transfected group. The cell viability was measured by CCK-8 assay; the expressions of p-tau and Aß was determined with ELISA Kit after AlCl(3) exposure or or/and ApoE ε4 transfection. Results: The viability of cells exposed to 400 µmol/L AlCl(3) or/and ApoE ε4 transfected were significantly lower than that of controls (P<0.05) . The expressions of total tau, tau-181, tau-231, tau-396 and Aß in 400 µmol/L Al(3)+ or/and ApoE ε4 transfected exposed cells showed significantly higher than those of controls (P<0.05) . Based on the factorial design, a significant interaction exists, and there is a synergistic effect between AlCl(3) and ApoE ε4 (P<0.05) . Conclusion: Aluminum and ApoE ε4 allele could increase expression of p-tau and Aß deposition; there was a synergistic interaction between aluminum and ApoE ε4 allele on cell death, tau phosphorylation and Aß deposition of SH-SY5Y.

[Influence of occupational aluminum exposure on cognitive function and glutamate receptor protein expression in workers].

Objective: To investigate the influence of occupational aluminum exposure on cognitive function and glutamate receptor protein expression in peripheral blood lymphocytes in workers and the possibility of glutamate receptor being used as a biomarker for cognitive impairment in aluminum workers. Methods: From October to December, 2014, cluster sampling was performed to select 121 workers in aluminum electrolysis workshop as exposure group and 231 workers in thermoelectric workshop and logistics department as control group. Mini-Mental State Examination, clock drawing test, digit span test (DST) , verbal fluency test (VFT) , and Fuld Object-Memory (FOM) Evaluation were used to analyze cognitive function. Graphite furnace atomic absorption spectrophotometry was used to measure plasma aluminum level as an exposure indicator. Enzyme-linked immunosorbent assay was used to measure the content of glutamate receptor proteins in peripheral blood lymphocytes, including the subunits of N-methyl-D-aspartate receptor NR1, NR2A, and NR2B and metabotropic glutamate receptor 1 (mGluR1) . The correlation between cognitive function indices and the content of glutamate receptor proteins was analyzed. Results: There was no significant difference in plasma aluminum level between the control group and the exposure group (132.52±80.40 µg/L vs 182.88±72.32 µg/L, P>0.05) . According to the plasma aluminum level, the study subjects were divided into control group and low-, medium-, and high-level plasma aluminum groups, and there were significant differences in plasma aluminum level between these groups (all P<0.01) . The high-level plasma aluminum group had a significantly lower memory ability score than the control group and the low- and medium-level plasma aluminum groups (all P<0.05) . The high-level plasma aluminum group had lower DST and digital span forward (DSF) scores than the control group and the low-and medium-level plasma aluminum groups. The low-, medium-, and high-level plasma aluminum groups had lower digital span backward (DSB) scores than the control group. The medium-and high-level plasma aluminum groups had lower VFT scores than the control group and the low-level plasma aluminum group. The high-level plasma aluminum group had significantly lower expression of NR1 and NR2A proteins than the control group and the low-and medium-level plasma aluminum groups, and the medium- and high-level plasma aluminum groups had significantly higher expression of mGluR1 protein than the control group and the low-level plasma aluminum group (all P<0.05) . The expression of NR1 and NR2A proteins was negatively correlated with plasma aluminum level (r=-0.475 and -0.692, both P<0.05) , andthe expression of mGluR1 protein was positively correlated with plasma aluminum level (r=0.756, P<0.05) . The expression of NR1 protein was positively correlated with DSF, DSB, DST, and VFT scores (r(s)=0.213, 0.249, 0.271, and 0.228, all P<0.05) , and the expression of NR2A protein was positively correlated with VFT score (r(s)=0.206, P<0.05) . Conclusion: Occupational aluminum exposure may affect workers' memory function, and the expression of NR1 and NR2A in peripheral blood lymphocytes is correlated with cognitive function indices and can be used as biomarkers for cognitive impairment in aluminum workers.

Expression of CD27, CD28 and IL-17A in peripheral blood from patients with colorectal carcinoma.

OBJECTIVE: To compare the different expressions of CD27, CD28, IL-17A, IFN-γ and TNF-α in the peripheral blood sampled from patients with colorectal carcinoma and healthy volunteers. PATIENTS AND METHODS: Vδ2 T cells were isolated from the peripheral blood mononuclear cells (PBMCs) of patients with the colorectal carcinoma (CRC, n = 30) and healthy controls (HC, n = 21). The proportion of CD27, CD28, IL-17A, IFN-γ and TNF-α of Vδ2 T cells was detected by the flow cytometry. RESULTS: We found that the proportion of IL-17A of Vδ2 T cells in PBMCs was higher in the CRC vs. the HC group (p < 0.05). A significant positive correlation was observed between the expression of IFN-γ and TNF-α of Vδ2 T cells. In the CRC patients, the proportions of IL-17A of CD27- Vδ2 T cells and CD28+ Vδ2 T cells were higher than those of CD27+ Vδ2 T cells and CD28- Vδ2 T cells, whereas the expression of IFN-γ and TNF-α of CD27-Vδ2 T cells was lower than that of CD27+ Vδ2 T cells. CONCLUSIONS: Vδ2 T cells from PBMCs had higher expression of IL-17A in CRC patients than that in the HC group. The expression of IFN-γ and TNF-α of Vδ2 T cells from PBMCs was positively correlated. The cytokine profiles of peripheral Vδ2 T cells were likely determined by a CD27 and CD28 involving mechanism.

[Association between H3K4me3/BDNF and the cognitive function of workers occupationally exposed to aluminum].

Objective: To investigate the influence of occupational aluminum exposure on cognitive function and its relationship with tri-methyl histone H3 lysine residues 4 points (H3K4me3) and brain-derived neurotrophic factor (BDNF) levels. Methods: By cluster random sampling method, a total of 235 cases of male workers selected from a Shanxi aluminum factory were recruited in the study in September 2015. Used the occupational epidemiological investigation questionnaire, which included Mini-Mental State Examination (MMSE) , Clock Drawing Test (CDT) , Digit Span Test (DST, including forward test DSFT and backward test DSBT) , Fuild Object Memory Evaluation (FOME) and Verbal Fluency Test (VFT) , to collect workers' basic information and assess their cognitive function score. Detected the concentration of aluminum in plasma by graphite furnace atomic absorption spectrometry. Workers were divided into three groups by the 25 percentile and 75 percentile of the aluminum content, such as low, middle and high aluminum concentration groups. The concentrations of H3K4me3 in lymphocyte and BDNF in plasma were determined by enzyme-linked immunosorbent assay. Results: The levels of aluminum in plasma was 134.36 (100.14, 178.96) µg/L. The scores of MMSE, DSFT, DSBT, DST of high aluminum concentration group were lower than low aluminum group (27.98±1.25 vs 28.83±1.54, 9.19±2.00 vs 10.64±2.87, 6.08±1.63 vs 7.19±3.07, 15.27±3.11 vs 17.81±4.72, all P<0.05) , the scores of CDT, FOME, VFT among three groups had no statistical significance (all P>0.05) . The expression levels of H3K4me3 and BDNF of high aluminum concentration group were lower than the low group [ (18.45±9.81) ng/µg Pro vs (23.76±9.89) ng/µg Pro, (26.07±10.18) ng/ml vs (31.66±9.24) ng/ml, all P<0.05]. Multiple correlation analysis showed that aluminum concentration were negatively correlated toH3K4me3, BDNF, MMSE, DSFT, DST, respectively (r(s)=-0.307、-0.214、-0.252、-0.197, -0.181, all P<0.01) . Conclusion: Exposure to occupational aluminum for a long time may change cognitive function, which go along with the decreasing of H3K4me3 level in lymphocyte and BDNF protein expression in plasma.

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly.

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.

The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis.

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.

Expression, purification, and activity of recombinant MHV-A59 3CLpro.

The 3C-like proteinase (3CLpro) of MHV-A59 is predicted to mediate the majority of proteolytic processing events within the gene 1 polyprotein. We have overexpressed 3CLpro in E. coli as a fusion protein with maltose binding protein (MBP). The MBP-3CLpro fusion protein was purified from contaminating E. coli proteins by amylose column chromatography, and r3CLpro was cleaved from the fusion protein by factor Xa. Recombinant 3CLpro (r3CLpro) was able to cleave a polypeptide substrate containing mutated inactive 3CLpro and portions of the flanking domains. R3CLpro cleaved substrate completely within 5 minutes and the activity of r3CLpro was sensitive to inhibition by serine and cysteine proteinase inhibitors; however, it was not inhibited by EDTA, suggesting that metal ions were not critical for 3CLpro activity.

Processing of the MHV-A59 gene 1 polyprotein by the 3C-like proteinase.

The 3C-like proteinase of mouse hepatitis virus (MHV-3CLpro) is predicted to cleave at least 10 sites in the gene 1 polyprotein, resulting in processing of proteinase, polymerase and helicase proteins from the polyprotein. We have used E. coli expressed recombinant 3CLpro (r3CLpro) to define cleavage sites in carboxy-terminal region of the ORF 1a polyprotein. Polypeptides containing one or more putative 3CLpro cleavage site were translated in vitro from subcloned regions of gene 1, and the polypeptides were incubated with r3CLpro. Analysis of the cleavage products confirmed several putative cleavage sites, as well as identifying cleavage sites not previously predicted by analysis of the MHV sequence. Antibodies directed against a portion of the ORF 1a polyprotein were used to probe virus infected cells, and detected proteins that correspond to the cleavage sites used by 3CLpro in vitro. These results suggest that MHV 3CLpro cleaves at least 7 sites in the ORF 1a polyprotein, and that the specificity of 3CLpro for cleavage site dipeptides may be broader than previously predicted.

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.