Annual prevalence and economic burden of genital warts in Korea: Health Insurance Review and Assessment (HIRA) service data from 2007 to 2015.

This study evaluated the annual prevalence of anogenital warts (AGW) caused by human papillomavirus (HPV) and analysed the trend in annual per cent changes (APC) by using national claims data from the Health Insurance Review and Assessment of Korea, 2007-2015. We also estimated the socio-economic burden and co-morbidities of AGW. All analyses were performed based on data for primary A63.0, the specific diagnosis code for AGW. The socio-economic cost of AGW was calculated based on the direct medical cost, direct non-medical cost and indirect cost. The overall AGW prevalence and socio-economic burden has increased during the last 9 years. However, the prevalence of AGW differed significantly by sex. The female prevalence increased until 2012, and decreased thereafter (APC + 3·6%). It would fall after the introduction of routine HPV vaccination, principally for females, in Korea. The male prevalence increased continuously over time (APC + 11·6%), especially in those aged 20-49 years. Referring to the increasing AGW prevalence and its disease burden, active HPV infection control surveillance and prevention in males are worth consideration.

A new surveillance indicator identifying optimal timeliness and accuracy: application to the Korean National Notifiable Disease Surveillance System for 2001-2007.

Although immediate notification of a case is crucial for epidemic control, clinicians may delay notification due to uncertainties in diagnosis, reflecting a trade-off between timeliness and the accuracy of surveillance. We assessed this trade-off for four epidemic-prone diseases that require immediate notification of suspected cases: shigellosis, typhoid fever, paratyphoid fever, and cholera in the Korean National Notifiable Disease Surveillance System data for 2001-2007. Timeliness was measured as the time to registration (T R), being the time interval from symptom onset to notification by the clinician to the local public health centre. We introduced a new index, 'time-accuracy trade-off ratio' to indicate time saved by clinical vs. laboratory-based notifications. Clinical notifications comprised 34.4% of total notifications, and these showed a shorter median T R than laboratory-based notifications (1-4 days). The trade-off ratio was greatest for shigellosis (3.3 days), and smallest for typhoid fever (0.6 days). A higher trade-off ratio provides stronger evidence for clinical notification without waiting for laboratory confirmation.

Effects of covered solid sorbent tube sample holders on organic vapor measurements.

A study was conducted to examine whether there are significant differences between organic vapor concentrations measured using charcoal tubes with three different configurations: uncovered sample holder (open tube), SKC, and Buck brand covered sample holders. A fractional factorial experimental design was used with the following factors and levels: vapor (n-hexane vs. m-xylene), pump type (pulsating vs. continuous), exposure profile (variable vs. constant), flow rate (30 mL/min vs. 200 mL/min), duration (30 min vs. 80 min), and sample placement (mannequin vs. free hanging). Two of each sampler configuration (six total) were placed in an exposure chamber, and a dynamic test-atmosphere generation system was used to prepare atmospheres containing approximately 12-15 ppm n-hexane or m-xylene with exposure profiles and sampling conducted according to a run sheet generated for the experimental design. A total of 24 runs were completed with six samplers per run, yielding 144 samples that were analyzed by gas chromatography/flame ionization detector. Concentration results for each pair of SKC and Buck covered sample holders were averaged and normalized by dividing by the average result for the open tube sampler from the same run to eliminate the effect of daily variation in chamber concentrations. The resulting ratio of covered sample tube holder and open tube concentrations was used as the response variable. Results of analysis of variance using the general linear model (MINITAB 16) identified statistically significant main effects and/or interactions for pump type, exposure profile, flow rate, and sample holder. However, the magnitude of the effects was generally less than 10%, and overall mean concentration ratios were 0.989 and 1.02 for the Buck and SKC sample holders, respectively. These results show good agreement between covered sample holder results and open tube measurements and demonstrate that exposure assessment errors resulting from the use of covered sorbent tube sample holders for organic vapor monitoring are relatively small (<10%) and not likely to be of practical importance.

Characterization of hydrophobic-ligand-binding proteins of Taenia solium that are expressed specifically in the adult stage.

Taenia solium, a causative agent of taeniasis and cysticercosis, has evolved a repertoire of lipid uptake mechanisms. Proteome analysis of T. solium excretory-secretory products (TsESP) identified 10 kDa proteins displaying significant sequence identity with cestode hydrophobic-ligand-binding-proteins (HLBPs). Two distinct 362- and 352-bp-long cDNAs encoding 264- and 258-bp-long open reading frames (87 and 85 amino acid polypeptides) were isolated by mining the T. solium expressed sequence tags and a cDNA library screening (TsHLBP1 and TsHLBP2; 94% sequence identity). They clustered into the same clade with those found in Moniezia expansa and Hymenolepis diminuta. Genomic structure analysis revealed that these genes might have originated from a common ancestor. Both the crude TsESP and bacterially expressed recombinant proteins exhibited binding activity toward 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), which was competitively inhibited by oleic acid. The proteins also bound to cis-parinaric acid (cPnA) and 16-(9-anthroyloxy) palmitic acid (16-AP), but showed no binding activity against 11-[(5-dimethylaminonaphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA) and dansyl-DL-α-aminocaprylic acid (DACA). Unsaturated fatty acids (FAs) showed greater affinity than saturated FAs. The proteins were specifically expressed in adult worms throughout the strobila. The TsHLBPs might be involved in uptake and/or sequestration of hydrophobic molecules provided by their hosts, thus contributing to host-parasite interface interrelationships.

Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models.

Identification of antigens that provide protective immunity via prophylactic and therapeutic vaccination against Mycobacterium tuberculosis is critical for the development of subunit vaccines for tuberculosis (TB). In this study, we performed a head-to-head comparison of seven well-known TB antigens delivered by DNA vaccine, and evaluated their respective immunogenicities and protective efficacies in pre- and post-exposure mouse models. All TB antigens were designed as a chimeric fusion with Flt3-L to enhance antigen-specific T-cell immunity upon vaccination. Prophylactic vaccination with the Flt3L (F)-Mtb32 DNA vaccine elicited significant protection in both the spleen and lungs against M. tuberculosis challenge, comparable to the Bacillus Calmette-Guerin vaccine. F-Ag85A and F-Mtb32 DNA vaccines, in combination with chemotherapy, reduced the bacterial burden to undetectable levels in the lungs of all mice infected with M. tuberculosis. These data collectively indicate that the F-Mtb32 DNA vaccine confers the most efficient protective immunity that suppresses bacterial growth in the active or latent status of M. tuberculosis.

Identification and biochemical characterization of two novel peroxiredoxins in a liver fluke, Clonorchis sinensis.

We identified 2 novel genes encoding different 2-Cys peroxiredoxins (PRxs), designated CsPRx2 and CsPRx3, in Clonorchis sinensis, which invades the human hepatobiliary tracts. The CsPRx2 gene expression was temporally increased along with the parasite's development and its protein product was detected in almost all parts of adult worms including subtegument, as well as excretory-secretory products. Conversely, CsPRx3 expression was temporally maintained at a basal level and largely restricted within interior parts of various tissues/organs. The recombinant forms of CsPRx proteins exhibited reducing activity against various hydroperoxides in the presence of either thioredoxin or glutathione (GSH) as a reducing equivalent, although they preferred H2O2 and GSH as a catalytic substrate and electron donor, respectively. A steady-state kinetic study demonstrated that the CsPRx proteins followed a saturable, Michaelis-Menten-type equation with the catalytic efficiencies (kcat/Km) ranging from 103 to 104 M-1 s-1, somewhat lower than those for other PRxs studied (104-105 M-1 s-1). The expression patterns and histological distributions specific to CsPRx2 and CsPRx3 might suggest different physiological functions of the antioxidant enzymes in protecting the worms against oxidative damage.

Serodiagnostic reliability of single-step enriched low-molecular weight proteins of Taenia solium metacestode of American and Asian isolates.

The low-molecular weight proteins (LMWPs) of Taenia solium metacestode (TsM) constituted pertinent serodiagnostic antigens for cysticercosis. We established a novel single-step purification of the LMWPs from TsM cyst fluid (CF). When the CF was precipitated with trichloroacetic acid/acetone mixture at the final concentrations of 5 and 50%, most LMWPs (ranging 7-38kDa) remained in the supernatant fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses revealed that the LMWPs consisted mainly with the previously reported subunit proteins of the 120- and 150-kDa antigen complexes. Serum samples from neurocysticercosis (NC) and other helminthic infections, as well as those from healthy controls were tested by both immunoblotting and micro-ELISA. In 135 cases of active stage NC patients, 132 cases (97.7%) showed positive reactions. Serum samples from other helminthic diseases (n=125) and healthy controls (n=100) exhibited no positive reactions except for cystic echinococcosis, of which 12% (3/25 cases) exhibited low levels of cross-reactivity. The LMWPs from different geographical regions (Korea and Mexico) showed diagnostic sensitivity and specificity of 97.7% and 98.7% against active stage NC. Our single-step separation method for the LMWPs provided excellent performance with easy applicability and high reproducibility, which has a great benefit for preparation of potent antigen in endemic areas.

Two novel phospholipid hydroperoxide glutathione peroxidase genes of Paragonimus westermani induced by oxidative stress.

SUMMARY: Phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We characterized 2 novel GPx genes from a lung fluke, Paragonimus westermani (designated PwGPx1 and PwGPx2). These single copy genes spanned 6559 and 12 371 bp, respectively, and contained each of 5 intervening introns. The PwGPx2 harboured a codon for Sec and a Sec insertion sequence motif. Proteins encoded by the Paragonimus genes demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic and glutathione-binding domains and absence of the subunit interaction domain. Expression of PwGPx1 increased gradually as the parasite matured, whereas that of PwGPx2 was temporally regulated. PwGPx2 was expressed at the basal level from the metacercariae to the 3-week-old juveniles; however, the expression was significantly induced in the 7-week-old immature worms and reached a plateau in the 12-week-old adults and eggs. PwGPx1 and PwGPx2 were largely localized in vitellocytes within vitelline glands and eggs. Oxidative stress-inducible paraquat, juglone and H2O2 substantially augmented the PwGPx1 and PwGPx2 expressions in viable worms by 1.5- to 11-fold. Our results strongly suggested that PwGPxs may actively participate in detoxification of oxidative hazards in P. westermani.

Nested PCR-based detection of Toxoplasma gondii in German shepherd dogs and stray cats in South Korea.

The prevalence of Toxoplasma gondii was surveyed by using a nested polymerase chain reaction (PCR) that was targeted to T. gondii B1 gene in German shepherd dogs and stray cats. Sixty-four (46.3%) out of 138 German shepherd dogs and 50 (47.2%) out of 106 stray cats were tested positive by the nested PCR assay, respectively. There was no significant difference in gender or age in German shepherd dogs and stray cats. In the five positive dogs and five positive cats, the nucleotide partial sequence of the T. gondii B1 gene was identified by direct sequence analysis. All the sequences were identical to each other and the corresponding sequence, T. gondii B1 gene (Accession No. AF179871). The results suggest that the prevalence of T. gondii is high, and the nested PCR assay is useful for early detection of T. gondii for asymptomatic dogs and cats.

Effects of culture conditions and nuclear transfer protocols on blastocyst formation and mRNA expression in pre-implantation porcine embryos.

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis (BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P<0.05), number of inner cell mass cells (13.3+/-4.3 vs. 7.6+/-2.2, P<0.05), and total cells (57.9+/-19.5 vs. 36.3+/-8.2, P<0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0mM calcium in fusion/activation medium (1.0C), and 7.5mug/mL cytochalasin B treatment (0.1C&CB) yielded higher rates (P<0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased (P<0.05) in 1.0C (77.4+/-28.9) compared to the control (58.5+/-22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes (BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

The clinical and immunogenetic characteristics of adult-onset type 1 diabetes mellitus in Korea.

Although the HLA class II alleles and immunological abnormalities are associated with type 1 diabetes mellitus (T1DM) in all racial groups, there are considerable variations in the genotypes and the prevalence of autoantibodies. In order to investigate the characteristics of the immunogenetic patterns and to use these as an early diagnostic tool and guideline for a therapeutic plan, we examined the clinical characteristics and the patterns of anti-GAD antibody (GADA), IA-2 antibody (IA-2A), HLA-DR and HLA-DQ in Korean adult-onset T1DM patients. Adult-onset patients had higher serum C-peptide levels than child-onset patients. In adult-onset patients, the prevalence of GADA and IA-2A were 59.5% and 15.3% respectively, and increased frequencies of HLADR4 and-DR9 were found. The frequencies of HLADQA1,-DQB1 and-DQ heterodimers were similar to those of the control, but child-onset patients had high frequencies of the HLA-DR3,-DR4,-DR9, DQA1*0301, DQA1*0501 and DQB1*0201 genotypes. In conclusion, Korean adult-onset T1DM patients had a lower prevalence of GADA, which was comparable to that found in Caucasian patients. The detection of GADA might help to predict the insulin dependency of adult-onset diabetes. Difference in the frequencies of diabetes associated with HLA type suggests that there might be a heterogeneity in the pathogenesis of diabetes according to the age of onset.

A heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, induced protective immunity against Toxoplasma gondii infection in mice.

SUMMARYThe dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-gamma) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.

An Internet-based surveillance system for tuberculosis in Korea.

SETTING: The Korea Tuberculosis Surveillance (KTBS) network includes 248 health centres throughout the country, as well as other public and private health institutions. OBJECTIVE: To develop a web-based surveillance system for tuberculosis (TB) and to monitor implementation of the National TB Control Programme (NTP) on an ongoing basis. DESIGN: A TB notification form was developed with new case definitions, and standardised to obtain uniform essential information of the cases with ease and speed. Data collection, compilation, analysis and feedback were made available at every level of the health authority via the Internet without restrictions of time and space. RESULTS: The Internet-based surveillance system was successfully implemented across the country, providing real-time national figures of TB using different variables-patient, time, area, site and type of disease--and facilitating on-line evaluation of NTP implementation. CONCLUSION: The web-based surveillance system has been well established within the existing health infrastructure, providing real-time figures on the TB burden. However, it requires continued improvement of the quality of information and of case reporting activities.

Urinary transforming growth factor-beta-induced gene-h3 (betaig-h3) as a sensitive predictor in chronic cyclosporine nephrotoxicity.

Transforming growth factor (TGF)-beta is involved in the pathogenesis of chronic cyclosporine nephrotoxicity (CyAN). Since the expression of TGF-beta induced gene h3 (betaig-h3) is up-regulated by TGF-beta, we evaluated the potential role of betaig-h3 as a sensitive urinary marker to monitor the progression/regression of chronic CyAN. Urinary betaig-h3 levels were determined using an enzyme-linked immunosorbent assay in nine patients with chronic CyAN and 13 patients with stable graft function. We scored the extent of tubulointerstitial fibrosis (TIF) and using immunoperoxidase labeling, determined betaig-h3 expression in renal tissues of patients with chronic CyAN. Urinary betaig-h3 excretion was higher in chronic CyAN compared to control subjects (173.4+/-26.0 vs 62.6+/-5.0 ng/mg creatinine, P<.01). In chronic CyAN, the degree of TIF correlated with increased urinary betaig-h3 levels (r=.785, P<.05). In kidneys with chronic CyAN, betaig-h3 labeling was more prominent at the basement membranes (BM) of the tubules where inflammatory cells had infiltrated the surrounding interstitium. Moreover, the BM of the atrophied tubules and their surrounding interstitium were strongly labeled. Urinary betaig-h3 levels decreased from 173.4+/-26.0 to 64.9+/-14.4 ng/mg creatinine at 1 month after discontinuation of CyA or reduction in CyA dosage (P<.01) despite unchanged serum creatinine levels. Urinary betaig-h3 levels increased in patients with chronic CyAN and decreased after discontinuation or reduction of CyA dosage. Our results suggested that urinary betaig-h3 levels could be used as a sensitive urinary marker to monitor the progression or regression of chronic CyAN.

Proteomic analysis of a 120 kDa protein complex in cyst fluid of Taenia solium metacestode and preliminary evaluation of its value for the serodiagnosis of neurocysticercosis.

Cyst fluid (CF) of Taenia solium metacestode (TsM) is an important source of serodiagnostic antigens. We have investigated the molecular characteristics of the 120 kDa protein complex in TsM CF purified by fast performance liquid chromatography. The structure of the purified protein was characterized by a variety of proteomic analyses. The protein was found to consist of 2 major components of 42-46 and 22-28 kDa, and shared 3 subunits of 14, 16 and 18 kDa. The 42-46 kDa component was determined to contain 3 additional subunits of 22, 28 and 38 kDa. These 6 subunits were shown to originate from either the 14 or 18 kDa precursor. We assessed the antibody reactivity of the native protein, its individual subunits and the recombinant 14 and 18 kDa proteins, and found that the 120 kDa protein, particularly 14 and 18 kDa subunits revealed high reliability for differentiation of active and mixed stage NC from chronic NC. The subunits of the 120 kDa protein complex identified herein represent some of the low-molecular weight glycoproteins which have been described in several previous studies. Recognizing and understanding the structural and immunological relationship of these proteins will facilitate the development of new serodiagnostic assays.

Feasibility of baculovirus-expressed recombinant 10-kDa antigen in the serodiagnosis of Taenia solium neurocysticercosis.

Bacterially expressed recombinant 10-kDa protein of Taenia solium metacestode (TsM) was previously found to be reliable in the diagnosis of active stage neurocysticercosis (NCC) by immunoblotting but not by ELISA. In this study, we evaluated the diagnostic feasibility of detecting eukaryote-expressed recombinant 10-kDa protein of TsM by ELISA (rTsM10-ELISA) in the serum and cerebrospinal fluid (CSF) from NCC patients. In 45 cases of active NCC, 91.1 and 97.8% cases showed positive reactions for serum and CSF by rTsM10-ELISA. ELISA employing the crude cyst fluid antigen (CF-ELISA) also revealed a similar result. Negligible cross-reactions were observed in serum samples from control subjects and from subjects with other helminthic diseases by rTsM10-ELISA (5/139 cases, 3.6%). By contrast, CF-ELISA demonstrated a high degree of cross-reactivity (24/139, 17.3%) especially from those patients with alveolar and cystic echinococcoses. The overall sensitivity and specificity of rTsM10-ELISA were 94.3 and 96.4%; and those of CF-ELISA were 95.7 and 84.5%, for serum and CSF, respectively. Antibody responses to rTsM10 were detected as early as 3 months after experimental infection of T. solium eggs in pigs. Our results show that ELISA with rTsM10 could be highly applicable in the serodiagnosis of NCC from early stage of infection.

Ambulatory treatment of multidrug-resistant pulmonary tuberculosis patients at a chest clinic.

SETTING: Retrospective cohort analysis of multidrug-resistant tuberculosis (MDR-TB) patients treated at a Korean National Tuberculosis Association out-patient chest clinic. OBJECTIVE: To evaluate treatment outcomes and contributing factors. DESIGN: A review of clinical records of 1011 pulmonary MDR-TB patients retreated with individualised regimens selected on the basis of previous chemotherapy and drug susceptibility testing from 1988 to 1996. RESULTS: The patients (mean age 38.6 years) had resistant organisms to an average of 3.7 drugs and were retreated with an average of 4.2 drugs which they had previously not taken and to which they were susceptible. Treatment outcomes were as follows: 487 cases (48.2%) cured, 82 (8.1%) failed, 394 (39.0%) defaulted, 45 (4.5%) transferred out, and three (0.3%) died. The treatment efficacy among those who completed chemotherapy was 85.6%. In a multivariate analysis favourable response was significantly associated with a greater number of newly prescribed drugs in the regimen to which they were susceptible (odds ratio [OR] 3.6; 95% confidence interval [CI] 1.3-9.5), younger age (OR 2.0; 95%CI 1.1-3.9), and a lower number of drugs to which they were resistant (OR 1.8; 95%CI 1.1-3.1). The case fatality rate, including the follow-up period, was 1.7% (17 cases). CONCLUSION: The cure rate of MDR-TB patients treated at an out-patient clinic was 48.2% due to a high defaulter rate (39.0%). However, 85.6% of those who completed treatment were cured.

Twenty-year trend of chronic excretors of tubercle bacilli based on the nationwide tuberculosis prevalence surveys in Korea, 1975-1995.

SETTING: A study of chronic excretors of tubercle bacilli (chronic cases) based on the nationwide random sample surveys of tuberculosis prevalence conducted in Korea from 1975 through 1995. OBJECTIVE: To investigate the temporal trend of the prevalence of chronic cases, and to match these with treatment outcomes and drug resistance rates. DESIGN: Bacillary cases were classified by history of chemotherapy into new (those who denied a history of chemotherapy), non-chronic (those who had taken chemotherapy for less than 2 years) and chronic cases (those who had taken chemotherapy for more than 2 years). RESULTS: Chronic cases decreased from 107 to 12 per 100000 population (annual rate of reduction [ARR] 11.89%) over the 20-year period. The ARR of chronic cases was significantly greater than that of new cases, and accelerated from 1985 (ARR 15.83%), after the application of short course chemotherapy. Rates of overall drug resistance rates increased up to 1980, and those of multidrug resistance up to 1985, followed by a decrease thereafter. A reduction in chronic cases was observed even during the period of increase in drug resistance (including multidrug resistance). CONCLUSION: The prevalence of chronic tuberculosis cases has decreased due to improvements in overall treatment outcome.

Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice.

A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappaB (NF-kappaB) activity and tumor necrosis factor (TNF)-mediated programmed cell death (PCD). TNF dramatically increases A20 messenger RNA expression in all tissues. Mice deficient for A20 develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely. A20-deficient cells fail to terminate TNF-induced NF-kappaB responses. These cells are also more susceptible than control cells to undergo TNF-mediated PCD. Thus, A20 is critical for limiting inflammation by terminating TNF-induced NF-kappaB responses in vivo.

Yeast three-hybrid screening of rous sarcoma virus mutants with randomly mutagenized minimal packaging signals reveals regions important for gag interactions.

We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MPsi) made using PCR amplification after cotransformation with GagDeltaPR protein into yeast cells. Colonies with low beta-galactosidase activity were analyzed to locate mutations in MPsi sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.