A W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.
Objective: The purpose of this study was to explore the effect of PERIOD3 (PER3) genotypes on circadian rhythmicity in flight cadets after militarized management. Methods: We performed a preliminary study in 146 newly enrolled male flight cadets. Venous blood samples were collected, and genotyping of PER3 (4/5) was determined by using PCR. The morningness-eveningness questionnaire (MEQ) survey was given to flight cadets upon enrollment and after militarized management for 24 months respectively. Comparison of frequency distribution of PER3 genotypes between cases and controls (120 well-matched civilians) was performed using the X2 test. We also compared the circadian rhythmicity upon enrollment and 24 months after enrollment in flight cadets, and analyzed the connection of changes in circadian clock with PER3 genotypes. Results: The frequency distribution of PER3 genotypes in flight cadets was not significantly different from that in controls subjects. MEQ survey results showed chronotype within flight cadet group varied widely at the two time-points: the moderately morning type (50%) and the neither type (41.1%) upon enrollment; the neither type (76.7%) and the moderately morning type (21.2%) 24 months after enrollment. The circadian rhythm of individuals with the PER3 (5/5) genotype showed no significant difference before and after 24 months of militarized management, whereas notable changes were found in individuals with the PER3 (4/4) genotype (n=116, X2=37.26, P < 0.001). Conclusion: In conclusion, we provide some evidence that circadian rhythm of flight cadets with the PER3 (5) allele are less likely to be affected compared to those with the PER3 (4) allele.
Circadian rhythmicity; PER3 gene; flight cadet; militarized management
Malignant mesotheliomas of the testis arise from the tunica vaginalis, formed from the evagination of the abdominal peritoneum into the scrotum. It is an extremely rare tumor representing 0.3% to 5% of all malignant mesotheliomas. We presented an interesting case of 68-year-old male with swelling and slightly painful in the right scrotum. Histologically, the lesion were composed of small tubular, microcystic, gland lined by flattened epithelioid cells and vague signet ring cells set in a myxofibrous stroma, which is resemblance to adenomatoid tumor. But the tumor cells showed significant atypical cytologic morphology and invaded into spermatic cord tissue, which indicated the diagnosis of malignant tumor. Immunohistochemistry study showed positive expression of CK, CK5/6, CK7, Calretinin, D2-40 and Vimentin which indicated the diagnosis of malignant mesothelioma. This case of mesothelioma should be classified as epithelial in type. To our knowledge, the mesothelioma of the tunica vaginalis testis with adenomatoid tumor-like microscopic features is very rare.
Mesothelioma; tunica vaginalis testis; histology; adenomatoid
Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.
Objective: The present study aims to identify the differently expressed microRNA (miRNA) molecules and target genes of miRNA in the immune tolerance (IT) and immune activation (IA) stages of chronic hepatitis B (CHB). Methods: miRNA expression profiles of peripheral blood mononuclear cells (PBMCs) at the IT and IA stages of CHB were screened using miRNA microarrays and authenticated using a quantitative real-time polymerase chain reaction (RT-PCR). Gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the significant functions and pathways of possible target genes of miRNAs. Assays of the gain and loss of function of the miRNAs were performed to verify the target genes in THP-1 cell lines. The luciferase reporter test was used on 293T cells as direct targets. Results: Significantly upregulated miR-548 and miR-4804 were observed in the miRNA microarrays and confirmed by RT-PCR in PBMCs at the IT and IA stages of CHB. GO and KEGG analysis revealed that MiR-548 and miR-4804 could be involved in numerous signaling pathways and protein binding activity. IFNγR1 was predicted as a target gene and validated as the direct gene of MiR-548. Significant negative correlation was found between the miR-548ah and mRNA levels of IFN-γR1 in CHB patients. Conclusions: The abnormal expression profiles of miRNA in PBMCs could be closely associated with immune activation of chronic HBV infection. miR-548, by targeting IFN-γR1, may represent a mechanism that can facilitate viral pathogenesis and help determine new therapeutic molecular targets.
chronic hepatitis B; microRNA; immune tolerance; immuneactivation
Solitary fibrous tumor (SFT) is rare mesenchymal neoplasm that has been originally and most often documented in the pleura. Recently, the ubiquitous nature of the SFT has been recognized with reports of involvement of numerous sites all over the body such as: upper respiratory tract, somatic tissue, mediastinum, head, and neck. Less than 10 cases SFT of breast have been reported. Herein, we presented a 52-year-old Asian female with SFT of breast, this tumor showed predominant malignant features. To our knowledge, SFT of breast with such malignant evidence is extremely rare.
Solitary fibrous tumor; morphology; immunochemistry
Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3′ processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.
Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.
We have reported that p120-catenin could regulate β-catenin transcription in lung cancer cells, but the specific mechanism is unclear.
Methods and Results
In this study, bisulfite sequencing PCR showed that the β-catenin promoter region in SPC-A-1 and LTEP-a-2 lung cancer cell lines has Kaiso binding sites sequences and CpG islands which may combine with Kaiso. The demethylating reagent 5-Aza-2′-deoxycytidine significantly upregulated β-catenin mRNA expression in lung cancer cell lines, whereas expression was significantly reduced following transfection with Kaiso. However, the upregulation of β-catenin mRNA expression after treatment with 5-Aza-2′-deoxycytidine was not reduced by subsequent transfection with Kaiso cDNA. Chromatin immunoprecipitation showed that, in lung cancer cell lines, methylated CpG-dinucleotides sequences combined with Kaiso and the Kaiso binding sites sequence did not. The capacity of Kaiso to combine with p120-catenin isoforms was confirmed by immunoprecipitation.
Based on these results, we concluded that Kaiso participates in the regulation by p120ctn of β-catenin mRNA expression in the lung cancer cell lines.
Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.
Breast capillary hemangioma is a type of benign vascular tumor which is rarely seen. Little is known about its presentation on dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we describe a case of suspicious breast lesion detected by DCE-MRI and pathologically confirmed as capillary hemangioma. Our case indicates that a small mass with a superficial location, clear boundary, and homogeneous enhancement on DCE-MRI indicates the possible diagnosis of hemangioma, whereby even the lesion presents a washout type curve.
Mammography; Ultrasound; Dynamic contrast-enhanced magnetic resonance imaging; Hemangioma
We previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear.
Four lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment.
Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells.
X-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor to predict radiosensitivity of the tumor.
Axin; Methylation; Proliferation; Invasiveness; Radiosensitivity
Adenocarcinoma of the rete testis is very rare. There is still little knowledge about its etiology and pathogenesis. Herein, we present a case of rete testis adenocarcinoma in a 36-year-old Chinese male. The tumor was predominantly composed of irregular small tubules and papillary structures with cuboidal or polygonal cells. In peripheral area of the tumor, the remaining normal rete testis and adenomatous hyperplasia of the rete testis could also be seen, indicating the possible relationship between adenomatous hyperplasia and adenocarcinoma. In addition, the patient underwent a left hydrocelectomy because of the existence of hydrocele 3 years ago. But, it is unclear whether hydrocele and hydrocelectomy is its cause or just the early clinical presentation of the adenocarcinoma.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6757609119625499
Adenocarcinoma; Rete testis; Adenomatous hyperplasia
Epithelioid hemangioendothelioma is a relatively uncommon lesion usually presenting in soft tissues. The occurrence in the mediastinum is exceptional rare. Histologically, this tumor is characterized by epithelioid cells with intracytoplasmic vacuoles in a hyalinized or mucinous stroma. Occasionally, spindle cells or osteoclast-like giant cells can be observed. Herein, we present a case of epithelioid hemangioendothelioma in a 38 year-old Chinese male. The tumor was predominantly composed of abundant spindle cells with marked atypia and scattered osteoclast-like giant cells reminiscent of malignant fibrous histiocytoma. The unusual histological appearance can pose a great diagnostic challenge. It may be easily misdiagnosed, especially if the specimen is limited or from fine-needle aspiration.
Epithelioid hemangioendothelioma; Mediastinum; Osteoclast-like giant cells; Malignant fibrous histiocytoma
Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.
The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.
Histone deacetylase (HDAC) plays an important role in the deacetylation of histone, which can alter gene expression patterns and affect cell behavior associated with malignant transformation. The aims of this study were to investigate the relationships between HDAC1, HDAC2, clinicopathologic characteristics, patient prognosis and apoptosis, to clarify the mechanism of upregulation of the Axis inhibitor Axin (an important regulator of the Wnt pathway) by X-radiation and to elucidate the effect of siRNA on radiation therapy of non-small cell lung cancer (NSCLC).
HDAC1 and HDAC2 expression levels were measured by immunohistochemistry and reverse transcription PCR. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and fluorescence activated cell sorting. BE1 cells expressing Axin were exposed to 2 Gy of X-radiation.
Expression of HDAC1 and that of HDAC2 were correlated, and significantly higher in NSCLC tissues than in normal lung tissues (P < 0.05). HDAC1 and HDAC2 expression was correlated with pTNM stage and negatively correlated with differentiation of NSCLC and apoptotic index (P < 0.05). The prognosis of patients with low expression of HDAC1 and HDAC2 was better than that of those with high expression. X-radiation and siRNA inhibited HDAC1 and HDAC2 expression in NSCLC cells and Axin levels were significantly higher in BE1 cells.
X-radiation and siRNA inhibit expression of HDAC1 and HDAC2, weaken the inhibitory effect of HDAC on Axin, upregulate Axin expression and induce apoptosis of lung cancer cells. Inhibition of HDAC1 and HDAC2 is a means of enhancing the radiosensitivity of NSCLC.
HDAC1; HDAC2; Axin; Lung neoplasm; Radiosensitization
Mucoepidermoid carcinoma is a common malignant epithelial tumor of salivary glands, but relatively rare in lung. The histological features of mucoepidermoid carcinoma of the lung are similar to its counterpart arising from the salivary glands. Here, we reported a special tumor that occurred in the medial segment of the right lower lobe in a 22-year-old man. This tumor exhibited typical features of mucoepidermoid carcinoma with 3 cell types: squamoid cells, mucin-secreting cells and cells of intermediate type. These 3 types of cells organized into cysts, nests, glands and solid patterns. Specially, the inner lining cells of some glandular structures were uniform cuboidal and hobnail-like, similar to the alveolar epithelial cells. Immunohistochemistry staining revealed that the inner lining cells of glandular structures were positive for thyroid transcription factor-1 and surfactant protein-B, used as markers of alveolar epithelial cells, and were negative for p63. These findings for the first time demonstrated a rare alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung.
The virtual slide(s) for this article can be found here:
Mucoepidermoid carcinoma; Lung neoplasm; Thyroid transcription factor-1; Surfactant protein-B
Angiomyolipoma (AML) is a rare mesenchymal neoplasm of the tumor, composed of a varying heterogeneous mixture of three tissue components: blood vessels, smooth muscle and adipose cells. Hepatic AML may demonstrate a marked histological diversity. We herein present one case of hepatic AML exhibiting prominent inflammatory cells in the background, which happened in a 61-year-old Chinese female patient, without signs of tuberous sclerosis. Histologically, the striking feature was the infiltration of numerous inflammatory cells in the background, including small lymphocytes, plasma cells, and eosnophils. The tumor cells were spindled and histiocytoid in shape, with slightly eosinophilic cytoplasm, and arranged along the vessels or scattered among the inflammatory background. Sinusoid structure was obviously seen in the tumor. Mature adipocytes and thick-walled blood vessels were focally observed at the boundaries between the tumor and surrounding liver tissues. The tumor cells were positive immunostaining for HMB-45, Melan-A, and smooth muscle actin. The inflammatory AML should be distinguished from other tumors with inflammatory background such as inflammatory myofibroblastic tumor and follicular dendritic cell tumor and deserves wider recognition for its occurrence as a primary hepatic tumor.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1828633072762370
Angiomyolipoma; Perivascular epithelioid cell; Inflammatory; Liver
Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1NL4-3 infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased Kms for dNTPs. In contrast, M184I/V resulted in an increased Km for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.
The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
HIV-1 subtype C infections account for over half of global HIV infections, yet the vast focus of HIV-1 research has been on subtype B viruses which represent less than 12% of the global pandemic. Since HIV-1 reverse transcriptase (RT) is a major target of antiviral therapy, and since differential drug resistance pathways have been observed among different HIV subtypes, it is important to study and compare the enzymatic activities of HIV-1 RT derived from each of subtypes B and C as well as to determine the susceptibilities of these enzymes to various RT inhibitors in biochemical assays.
Recombinant subtype B and C HIV-1 RTs in heterodimeric form were purified from Escherichia coli and enzyme activities were compared in cell-free assays. The efficiency of (-) ssDNA synthesis was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Processivity was assayed under single-cycle conditions using both homopolymeric and heteropolymeric RNA templates. Intrinsic RNase H activity was compared using 5'-end labeled RNA template annealed to 3'-end recessed DNA primer in a time course study in the presence and absence of a heparin trap. A mis-incorporation assay was used to assess the fidelity of the two RT enzymes. Drug susceptibility assays were performed both in cell-free assays using recombinant enzymes and in cell culture phenotyping assays.
The comparative biochemical analyses of recombinant subtype B and subtype C HIV-1 reverse transcriptase indicate that the two enzymes are very similar biochemically in efficiency of tRNA-primed (-) ssDNA synthesis, processivity, fidelity and RNase H activity, and that both enzymes show similar susceptibilities to commonly used NRTIs and NNRTIs. Cell culture phenotyping assays confirmed these results.
Overall enzyme activity and drug susceptibility of HIV-1 subtype C RT are comparable to those of subtype B RT. The use of RT inhibitors (RTIs) against these two HIV-1 enzymes should have comparable effects.