Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Activin A/ACVR2A axis inhibits epithelial-to-mesenchymal transition in colon cancer by activating SMAD2.

Colorectal cancer is one of the most common malignancies worldwide. Liver metastasis is the major direct cause of colorectal cancer-related deaths. Although radical resection is the most effective treatment for colorectal cancer liver metastasis, several patients are not eligible for surgery. Therefore, there is a need to develop novel treatments based on the understanding of the biological mechanisms underlying liver metastasis in colorectal cancer. This study demonstrated that activin A/ACVR2A inhibits colon cancer cell migration and invasion, as well as suppresses the epithelial-to-mesenchymal transition of mouse colon cancer cells. This finding has been further validated in animal experiments. Mechanistic studies revealed that activin A binds to Smad2 (instead of Smad3) and activates its transcription. Analysis of the paired clinical samples further confirmed that the expression levels of ACVR2A and SMAD2 were the highest in adjacent healthy tissues, followed by primary colon cancer tissues and liver metastasis tissues, suggesting that ACVR2A downregulation may promote colon cancer metastasis. Bioinformatics analysis and clinical studies demonstrated that ACVR2A downregulation was significantly associated with liver metastasis and poor disease-free and progression-free survival of patients with colon cancer. These results suggest that the activin A/ACVR2A axis promotes colon cancer metastasis by selectively activating SMAD2. Thus, targeting ACVR2A is a potential novel therapeutic strategy to prevent colon cancer metastasis.

Comparative neutralizing potencies of antibodies suggest conservation as well as mechanistic differences in human cytomegalovirus entry into epithelial and endothelial cells.

Antibody neutralization of cytomegalovirus (CMV) entry into diverse cell types is a key consideration for development of vaccines and immunotherapeutics. CMV entry into fibroblasts differs significantly from entry into epithelial or endothelial cells: fibroblast entry is mediated by gB and gH/gL/gO, whereas both epithelial and endothelial cell entry require an additional pentameric complex (PC) comprised of gH/gL/UL128/UL130/UL131A. Because PC-specific antibodies in CMV-seropositive human sera do not affect fibroblast entry but potently block entry into epithelial or endothelial cells, substantially higher neutralizing potencies for CMV-positive sera are observed when assayed using epithelial cells as targets than when using fibroblasts. That certain sera exhibit similar discordances between neutralizing potencies measured using epithelial vs. endothelial cells (Gerna G. et al.J Gen Virol, 89:853-865, 2008) suggested that additional mechanistic differences may also exist between epithelial and endothelial cell entry. To further explore this issue, neutralizing potencies using epithelial and endothelial cells were simultaneously determined for eight CMV-positive human sera, CMV-hyperimmune globulin, and a panel of monoclonal or anti-peptide antibodies targeting specific epitopes in gB, gH, gH/gL, or the PC. No significant differences were observed between epithelial and endothelial neutralizing potencies of epitope-specific antibodies, CMV-hyperimmune globulin, or seven of the eight human sera. However, one human serum exhibited a six-fold higher potency for neutralizing entry into epithelial cells vs. endothelial cells. These results suggest that epitopes exist that are important for epithelial entry but are less critical, or perhaps dispensable, for endothelial cell entry. Their existence should be considered when developing monoclonal antibody therapies or subunit vaccines representing limited epitopes.

Levels of human cytomegalovirus miR-US25-1-5p and miR-UL112-3p in serum extracellular vesicles from infants with HCMV active infection are significantly correlated with liver damage.

Human cytomegalovirus (HCMV)-encoded microRNAs (miRNAs) are involved in posttranscriptional regulation of gene expression. Extracellular vesicles (EVs) can incorporate miRNAs. Relationship between HCMV infection and miRNAs in EVs remains unknown. EVs were isolated from supernatants of human embryonic lung fibroblasts (HELF) cells. Profiles of miRNAs in EVs were analyzed by deep sequencing. Dynamics of candidate viral miRNAs transportation via EVs was investigated using TaqMan PCR. Levels of candidate viral miRNAs in serum EVs from infants with HCMV active infection were detected and analyzed with their clinical index levels. A total of 16 HCMV miRNAs were found in EVs from infected HELF. Levels of miR-US25-1-5p and miR-UL112-3p in EVs increased at 6 h post-infection and were correlated with those in cells (for miR-US25-1-5p: r2 = 0.9375, p value < 0.05; for miR-UL112-3p: r2 = 0.7557, p value < 0.05). Viral miRNAs were transported into recipient cells at 2 h post-incubation. Moreover, levels of miR-US25-1-5p in serum EVs showed positive correlations with serum levels of γ-glutamyl transpeptidase, direct bilirubin, and total bile acid. Levels of miR-UL112-3p in serum EVs showed a positive correlation with serum levels of direct bilirubin. HCMV miRNAs could be transported to uninfected cells via EVs. Levels of miR-US25-1-5p and miR-UL112-3p in serum EVs from infants with HCMV active infection were significantly correlated with liver damage.

Characterization of Human Cytomegalovirus UL16 and UL17 Transcripts.

Up to now, the UL16-17 region of human cytomegalovirus (HCMV) has not been well characterized at the level of mRNA and protein, especially for the Han strain, the first clinical HCMV strain in China. In previous studies, three transcripts were detected from the UL16-17 region by northern blot analysis for Merlin strain. Transcriptions of UL16 and UL17 were also studied by 5' rapid amplification of cDNA ends (5'RACE) and deep sequencing for AD169 and Towne strains, respectively. However, details of 3' end of UL16 and UL17 transcripts have never been confirmed by 3'RACE. The expressing phage of the UL16-17 region needs further research by northern blot, too. In the present study, cDNA library screening, northern blot and RACE were used to identify the transcription characteristics of the UL16-17 region. Mainly, 3 clusters of transcripts with the same 3' end were found to be expressed from the UL16-17 region in both Han and AD169 strains. The lengths of the core transcripts among the 3 clusters were 1,254nt, 718nt and 468nt, respectively. The corresponding 5' ends are at nt23119, nt23655, nt23905 in the HCMV Han genome. The consistent 3' end is located at nt24372 in the Han genome. The 1,254nt and 468nt transcripts are transcribed in early and late phases, and the 718nt transcript is transcribed only in the late phase.

Automated Vulnerability Discovery and Exploitation in the Internet of Things.

Recently, automated software vulnerability detection and exploitation in Internet of Things (IoT) has attracted more and more attention, due to IoT's fast adoption and high social impact. However, the task is challenging and the solutions are non-trivial: the existing methods have limited effectiveness at discovering vulnerabilities capable of compromising IoT systems. To address this, we propose an Automated Vulnerability Discovery and Exploitation framework with a Scheduling strategy, AutoDES that aims to improve the efficiency and effectiveness of vulnerability discovery and exploitation. In the vulnerability discovery stage, we use our Anti-Driller technique to mitigate the "path explosion" problem. This approach first generates a specific input proceeding from symbolic execution based on a Control Flow Graph (CFG). It then leverages a mutation-based fuzzer to find vulnerabilities while avoiding invalid mutations. In the vulnerability exploitation stage, we analyze the characteristics of vulnerabilities and then propose to generate exploits, via the use of several proposed attack techniques that can produce a shell based on the detected vulnerabilities. We also propose a genetic algorithm (GA)-based scheduling strategy (AutoS) that helps with assigning the computing resources dynamically and efficiently. The extensive experimental results on the RHG 2018 challenge dataset and the BCTF-RHG 2019 challenge dataset clearly demonstrate the effectiveness and efficiency of the proposed framework.

Serologic and viral genome prevalence of HSV, EBV, and HCMV among healthy adults in Wuhan, China.

The worldwide infection rate of herpesvirus is high, but the detailed prevalence in China, especially the central area, remains unclear. In the present study, the prevalence of herpes simplex virus (HSV), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV) was investigated in 303 healthy adults in Wuhan, a representative city in Central China. Viral-specific IgG and IgM titers were examined in the serum by chemiluminescent immunoassay, and the existence of viral genomic DNA in blood cells was determined by nested PCR. The overall IgG seroprevalences were 81.5%, 95.4%, and 93.7% for HSV, EBV, and HCMV, while the corresponding IgM seroprevalences were only 6.3%, 2.3%, and 0. The viral genomic DNA of HSV, EBV, and HCMV was identified in the blood samples of 5.9%, 14.2%, and 22.8% of the tested donors, respectively. Significantly, less HSV IgM-positive samples were found in the population over 20 years old than below 20 group; female displayed higher chances for HSV IgG and genome positivity; and occupations such as waiters and medical staffs were shown to be with higher risk for HCMV genome positivity. This study provided useful reference data for the HSV, EBV, and HCMV prevalence in central China, and suggested the potential importance of detecting viral genome to complement serum test data.

Viral load and integration status of HPV58 associated with cervical lesion severity in women of Northeast China.

Objective: We aimed to evaluate the viral load and integration status of human papillomavirus 58 in women with different grades of cervical lesions to determine whether viral load and integration status are related to malignant transformation in HPV58-infected women. Methods: A total of 212 cervical specimens were collected from women in Northeast China who had undergone human papillomavirus genotyping and were HPV58-positive. The HPV58 viral load was determined using real-time polymerase chain reaction, and the integration status was discriminated using the ratio of HPV58 E2 gene copy number to E6 gene copy number. Results: The median HPV58 viral load in women with normal cervix or cervicitis, low-grade squamous cell intraepithelial lesion, high-grade squamous cell intraepithelial lesion and cervical cancer was 352.12, 864.21, 1199.75 and 693.04 copies/genome, respectively. High significance was obtained when comparing the viral load of infected women presenting normal/cervicitis with that of the women either with precancerous cervical lesions or cervical cancer (P < 0.05). The HPV58 genome was in the episomal form in 35 samples (16.5%), mixed episomal and integrated forms in 165 (77.8%) samples, and completely integrated into the host genome in 12 (5.7%) samples. The HPV58 E2/E6 copy number ratio in the cervical cancer group was significantly lower than that in the other groups (P < 0.01). Conclusions: The HPV58 viral load in patients with precancerous cervical lesions or cervical cancer increases significantly with disease progression. The HPV58 E2/E6 copy number ratio in patients with cervical cancer is lower than that for less severe cervical lesions, suggesting a high degree of viral integration may be a considerable risk factor for cervical cancer.

Multiple human papillomavirus infections and type-competition in women from a clinic attendee population in China.

To investigate the multi-infection patterns and type competition for human papillomavirus (HPV) in Chinese clinic attending women and evaluate the association between the infection status and cancer risk. Three hundred and thirty-two HPV-DNA-positive samples were genotyped for 21 HPVs and tested for 13 types of HPV neutralizing antibody using pseudoviron-based assay. Odds ratios (ORs) were calculated to evaluate the coinfection patterns for both DNA and neutralizing antibody (NAb), and the associations of HSIL+ with HPV DNA and NAb. Of the 332 HPV-DNA-positive subjects, 279 (84.0%) were detected as NAb positive. Multi-positive results were identified in 23.2% (77/332) HPV DNA tests and 60.2% (168/279) HPV NAb assays. The NAb titers for the multiple-positive samples (geometric mean 214) were significantly higher than the single-positive samples (geometric mean 114) (P < 0.01). HPV16-HPV52 was identified as a type-competition pair for both HPV DNA (OR = 0.09; 95%CI = 0.02-0.42) and NAb (OR = 0.27; 95%CI = 0.11-0.69) data. Compared to other types, HPV16 DNA was associated significantly with high risk of HSIL+ (OR = 2.57; 95%CI = 1.23-5.38). HPV16-HPV52 was identified as a potential type-competition pair, which might result in the type modulation with the implementation of the approved bi- or quadri-valent vaccines. J. Med. Virol. 88:1989-1998, 2016. © 2016 Wiley Periodicals, Inc.

Identification and BAC construction of Han, the first characterized HCMV clinical strain in China.

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects, and may lead to severe or lethal diseases in immunocompromised individuals. Several HCMV strains have been identified and widely applied in research, but no isolate from China has been characterized. In the present study, we isolated, characterized and sequenced the first Chinese HCMV clinical strain Han, and constructed the novel and functional HCMV infectious clone Han-BAC-2311. HCMV Han was isolated from the urine sample of a Chinese infant with multiple developmental disorders. It expresses HCMV specific proteins and contains a representative HCMV genome with minor differences compared to other strains. By homologous recombination using mini-F derived BAC vector pUS-F6, the infectious clone Han-BAC-2311 was constructed containing representative viral genes across the HCMV genome. The insertion site and orientation of BAC sequence were confirmed by restriction enzyme digestion and Southern blotting. The reconstituted recombinant virus HanBAC-2311 expresses typical viral proteins with the same pattern as that of wild-type Han, and also displayed a similar growth kinetics to wild-type Han. The identification of the first clinical HCMV strain in China and the construction of its infectious clone will greatly facilitate the pathogenesis studies and vaccine development in China.

A cluster of 3' coterminal transcripts from US12-US17 locus of human cytomegalovirus.

Among all the human cytomegalovirus (HCMV) gene families, US12 family is relatively undefined in their transcriptional profile and biological functions. In this study, the transcription pattern and characteristics of HCMV US12-US17 gene region were studied extensively. Twenty-three clones harboring US12 cDNA sequence were screened out from a late cDNA library of an HCMV clinical isolate, Han. Using a set of US12-US17 gene-specific probes, six transcripts from US12-US17 locus were detected by northern blot at late kinetics of the clinical isolate. One additional transcript was found in late RNA of HCMV strain AD169. No evidence showing these transcripts contain introns by reverse transcription PCR. 3' and 5' termini of these transcripts were confirmed by Rapid Amplification of cDNA Ends. A novel protein-coding region was predicted in the shorter US14 transcript with an alternative in-frame 5' translation initiation site compared to that of the previously predicted US14 ORF. Our findings demonstrate the existence of a cluster of 3' coterminal unspliced transcripts with distinct 5' transcriptional initiation sites originated from US12-US17 gene region in the late infection phase of an HCMV clinical strain.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemoperfusion for the treatment of gastric cancer: A single-centre retrospective study.

AIM: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemoperfusion (HIPEC) is the treatment regime most likely to achieve prolonged survival in patients with peritoneal carcinomatosis from gastroenteric cancer. To date, few publications have focused on the treatment of patients with gastric cancer alone. Several controversies remain unsolved, including the safety and effectiveness of the CRS-HIPEC combination regime, particularly in cases where HIPEC is used as adjuvant treatment after CRS. Therefore, in the current study, we aimed to evaluate the safety and effectiveness of CRS combined with HIPEC in patients with gastric cancer. METHOD: Data from 231 patients with a median age of 55.1 years treated with the CRS-HIPEC combination regime between January 2009 and December 2014 were retrospectively reviewed. All patients underwent the combination therapy (mean of 2.4 cycles per patient, range, 1 to 4 cycles). RESULTS: Median overall survival was 37.0 months, with 1-, 2- and 3-year survival rates recorded as 83.4%, 68.5%, and 38.7%, respectively. The serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) were significantly decreased after combination therapy in the completeness of cytoreduction (CCR)-0 and CCR-1 groups, while no significant changes observed in marker levels were observed in the CC ≥2 group. The post-operative morbidity and mortality rates were 6.9% and 0.9%, respectively. Multivariate analysis revealed low TNM tumour stage, ascites condition and CCR score as independent predictors for better survival. CONCLUSION: In view of the acceptable morbidity and mortality rates we propose that CRS combined with HIPEC presents an effective and safe treatment modality for patients with gastric cancer, especially in cases where optimal cytoreduction is achieved before the HIPEC procedure.

Thermo-chemotherapy Induced miR-218 upregulation inhibits the invasion of gastric cancer via targeting Gli2 and E-cadherin.

Thermo-chemotherapy has been proven to reduce the invasion capability of cancer cells. However, the molecular mechanism underlying this anti-invasion effect is still unclear. In this study, the role of thermo-chemotherapy in the inhibition of tumor invasion was studied. The results demonstrated that expression of miR-218 was downregulated in gastric cancer tissues, which had a positive correlation with tumor invasion and metastasis. In vitro thermo-chemotherapy increased miR-218 expression in SGC7901 cells and inhibited both proliferation and invasion of cancer cells. Gli2 was identified as a downstream target of miR-218, and its expression was negatively regulated by miR-218. The thermo-chemotherapy induced miR-218 upregulation was also accompanied by increasing of E-cadherin expression. In conclusion, the present study indicates that thermo-chemotherapy can effectively decrease the invasion capability of cancer cells and increase cell-cell adhesion. miR-218 and its downstream target Gli2, as well as E-cadherin, participate in the anti-invasion process.

Development of a fluorescence-based multiplex genotyping method for simultaneous determination of human papillomavirus infections and viral loads.

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing intraepithelial lesion or malignancy (NILM). The aims of the current study is to establish a method named BioPerfectus Multiplex Real Time (BMRT) HPV assay for simultaneous typing and quantifying HPVs, and to evaluate it by comparison with HPV GenoArray test and PCR-sequencing method, as well as histological status. METHODS: A total of 817 cervical specimens were evaluated by BMRT method and HPV GenoArray test, using PCR-sequencing method as the reference standard; simultaneously, high-risk HPV-16 and -18 DNA loads were assessed in 443 specimens to investigate the correlation with infection outcomes. RESULTS: The overall detection coincidence rate between BMRT assay and HPV GenoArray test is 96.6 % and the Kappa value is 0.760. In addition, the sensitivity and positive predictive value of BMRT is 98.4 % and 95.7 % compared with the results detected by PCR-sequencing method, respectively. HPV-16 viral load has a correlation with CINs or worse lesions. By comparing with infected women presenting NILM /cervicitis, the cutoff value for HPV-16 from patients with CINs was 0.827. With this cutoff value, 74.6 % sensitivity and 72.5 % specificity for prediction of HPV-16 infected patients with CINI and higher CIN were achieved. High significance was obtained when comparing the infected women presenting NILM/cervicitis with women either with CIN and cervical carcinomas (p < 0.001). CONCLUSIONS: The BMRT assay seemed to be a good alternative approach for HR-HPV testing, due to its high level of automation and ability to quantify HPV-16, HPV-18 and other HR-HPVs.

Human cytomegalovirus miR-UL36-5p inhibits apoptosis via downregulation of adenine nucleotide translocator 3 in cultured cells.

Human cytomegalovirus (HCMV) encodes at least 26 microRNAs (miRNA). These miRNAs are utilized by HCMV to regulate its own genes as well as the genes of the host cell during infection. It has been reported that a cellular gene, solute carrier family 25, member 6 (SLC25A6), which is also designated adenine nucleotide translocator 3 (ANT3), was identified as a candidate target of hcmv-miR-UL36-5p by hybrid PCR. In this study, ANT3 was further demonstrated to be a direct target of hcmv-miR-UL36-5p by luciferase reporter assays. The expression level of ANT3 protein was confirmed, by western blotting, to be directly downregulated by overexpression of hcmv-miR-UL36-5p in HEK293 cells, U373 cells and HELF cells. Moreover, HCMV-infected cells showed a decrease in the ANT3 protein level. Using ANT3-specific small interfering RNA (siRNA) and an inhibitor for hcmv-miR-UL36-5p, it was shown that inhibition of apoptosis by hcmv-miR-UL36-5p in these cells specifically occurred via inhibition of ANT3 expression. These results imply that hcmv-miR-UL36-5 may play the same role during actual HCMV infection in order to establish a balance between the host cell and the virus.

Characterization of a novel group of antisense transcripts in human cytomegalovirus UL83 gene region.

The rapid advances in research on antisense transcripts are gradually changing our understanding of the expression of the Herpesviridae genome. In this study, the transcripts of the human cytomegalovirus (HCMV) UL83 antisense strand were investigated in three clinical isolates. Three cDNA clones containing sequences with an antisense orientation to the UL83 gene were identified in a late HCMV cDNA library. The UL83 antisense transcripts (UL83asts) were then shown to be transcribed only in the late infection phase of the three clinical HCMV strains, using rapid amplification of cDNA ends (RACE) and northern blotting. These UL83asts were identical at their 3' termini but different at 5' ends. Two open reading frames were predicted in the UL83asts.

Case of Reye's syndrome accompanied by hemolytic anemia and cardiac injury after cytomegalovirus infection.

A 6-month-old girl with active human cytomegalovirus (HCMV) infection developed Reye's syndrome after vaccination. She suffered from uncommon complications of HCMV infection, including Coombs-negative hemolytic anemia and cardiac injury, but recovered after the appropriate treatment with prompt ganciclovir and symptomatic support. However, the patient died later as a result of a viral upper respiratory tract infection, which aggravated the primary disease. This case suggests that HCMV infection might be a causative agent of Reye's syndrome.

Genetic variations of E6 and long control region of human papillomavirus type 16 from patients with cervical lesion in Liaoning, China.

BACKGROUND: High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. Previous studies suggest that polymorphisms in the E6 gene or the long control region(LCR)of HPV16 may alter the oncogenic potential of the virus. The aims of this study were to investigate the genetic variations of HPV16 E6 gene and LCR in isolates from Chinese population and correlation of the E6 and LCR polymorphisms with disease status of infected patients. METHODS: HPV16 positive endocervical specimens were collected from 304 women living in Northeast of China. Sequences of E6 gene and LCR were analyzed by PCR-sequencing. RESULTS: Two lineages were found in the populations, including EUR lineage and As lineage. Based on the HPV16 prototype, the most frequent variation in the E6 gene was T178A/G (48.7%), followed by mutations of G94A (12.2%) and T350G (9.9%). The rank orders of incidence of E6 variations in amino acid were as follows: D25E (46.3%), L83V (9.9%) and H78Y (4.3%). Nucleotide variations in LCR were found in all the 304 isolates from HPV16 positive cervical samples. The most commonly observed LCR variations were the transition replacement G7193T, 7434CIns, G7521A and 7863ADel (100%). The As lineage was associated with HPV persistent infections and with disease status of ≥CIN2,3. The EUR lineage variants showed a negative trend of association with the severity of ≥CIN2,3. Among 41 variations found in LCR, 25 (61.0%) were located at the binding sites for transcription factors. Occurrence of ≥CIN2,3 was significantly associated with the mutations of R10G/L83V in E6 and the C7294T co-variation in LCR, after adjusting for ages of infected patients. CONCLUSIONS: Associations between As lineage and HPV persistent infections, and with disease status of ≥CIN2,3, and an association between the EUR lineage and negative trend of association with the severity of ≥CIN2,3 were found in this study. An association between a co-variation of R10G/L83V in E6 and C7294T in LCR and an increased risk for developing CIN-2,3 was found in a HPV16 infected population of Chinese women. These findings indicate that HPV16 polymorphism influences development of CIN-2,3.

The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1.

BACKGROUND: Interleukin-32 (IL-32) is an important factor in innate and adaptive immune responses, which activates the p38MAPK, NF-kappa B and AP-1 signaling pathways. Recent reports have highlighted that IL-32 is regulated during viral infection in humans. METHODS: Enzyme-linked immunosorbent assays (ELISA) were carried out to detect IL-32 levels in serum samples. Detailed kinetics of the transcription of IL-32 mRNA and expression of IL-32 protein during human cytomegalovirus (HCMV) infection were determined by semi-quantitative RT-PCR and western blot, respectively. The expression levels of hcmv-miR-UL112-1 were detected using TaqMan® miRNA assays during a time course of 96 hours. The effects of hcmv-miR-UL112-1 on IL-32 expression were demonstrated by luciferase assay and western blot, respectively. RESULTS: Serum levels of IL-32 in HCMV-IgM positive patients (indicating an active HCMV infection) were significantly higher than those in HCMV-IgM negative controls. HCMV infection activated cellular IL-32 transcription mainly in the immediately early (IE) phase and elevated IL-32 protein levels between 6 and 72 hours post infection (hpi) in the human embryonic lung fibroblast cell line, MRC-5. The expression of hcmv-miR-UL112-1 was detected at 24 hpi and increased gradually as the HCMV-infection process was prolonged. In addition, it was demonstrated that hcmv-miR-UL112-1 targets a sequence in the IL-32 3'-UTR. The protein level of IL-32 in HEK293 cells could be functionally down-regulated by transfected hcmv-miR-UL112-1. CONCLUSIONS: IL-32 expression was induced by active HCMV infection and could be functionally down-regulated by ectopically expressed hcmv-miR-UL112-1. Our data may indicate a new strategy of immune evasion by HCMV through post-transcriptional regulation.

Analysis and mapping of a 3' coterminal transcription unit derived from human cytomegalovirus open reading frames UL30-UL32.

BACKGROUND: It has been predicted that the UL31 gene originates from the positive strand of the human cytomegalovirus (HCMV) genome, whereas the UL30 and UL32 genes originate from the complementary strand. Except for the UL32 gene, the transcription of this gene region has not been investigated extensively. METHODS: Northern blotting, cDNA library screening, RACE-PCR,and RT-PCR were used. RESULTS: At least eight transcripts of the antisense orientation of UL31 were transcribed from the UL30-UL32 region during the late phase of HCMV infection. The 3' coterminus of these transcripts was located within the predicted UL30 gene. The longest 6.0-kb transcript was initiated upstream of the predicted UL32 gene. Other transcripts were derived from the predicted UL30 and UL31 gene region. Except for the previously predicted UL32 open reading frame (ORF), three novel ORFs, named UL31anti-1, UL31anti-2 and UL31anti-3, were located in the transcripts from the UL31anti-UL32 transcription unit. No transcription was found in UL31. CONCLUSION: A family of novel 3' coterminal transcripts was transcribed from the UL30-UL32 gene region.