Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/β dimer. Although the amount of the mutant hCGβ assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ∼50% of secreted β-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGβ genes CGB5 and CGB8

The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

The human osteosarcoma cell line U2OS is useful for studying genome replication of human papillomavirus (HPVs) subtypes that belong to different phylogenetic genera. In this study, we defined the HPV18 transcription map in U2OS cells during transient replication, stable maintenance and vegetative amplification by identifying viral promoter regions, transcription polyadenylation and splicing sites during HPV18 genome replication. Mapping of the HPV18 transcription start sites in U2OS cells revealed five distinct promoter regions (P102, P520, P811, P1193 and P3000). With the exception of P3000, all of these regions have been previously identified during productive HPV18 infection. Collectively, the data suggest that U2OS cells are suitable for studying the replication and transcription properties of HPVs and to serve as a platform for conducting high-throughput drug screens to identify HPV replication inhibitors. In addition, we have identified mRNA species that are initiated from the promoter region P3000, which can encode two E2C regulator proteins that contain only the C-terminal hinge and DNA-binding and dimerization domains of E2. We show that these proteins regulate the initial amplification of HPV18 by modulating viral transcription. Moreover, we show that one of these proteins can act as a transcriptional activator of promoter P102

A DNA Polymerase V Homologue Encoded by TOL Plasmid pWW0 Confers Evolutionary Fitness on Pseudomonas putida under Conditions of Environmental Stress

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P. putida. When rulAB-carrying cells from an 8-day-old culture were mixed with Pol V-negative cells from a 1-day-old culture, cells derived from the aged culture out-competed cells from the nonaged culture and overtook the whole culture. At the same time, bacteria from an aged culture lacking the rulAB genes were only partially able to out-compete cells from a fresh overnight culture of the parental P. putida strain. Thus, in addition to conferring resistance to DNA damage, the plasmid-encoded Pol V genes significantly increase the evolutionary fitness of bacteria during prolonged nutritional starvation of a P. putida population. The results of our study indicate that RecA is involved in the control of expression of the pWW0-encoded Pol V

Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385.

<p><b>A:</b> Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 <i>wt</i>, E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included <b>B:</b> U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C_N). The value obtained from the HPV18 <i>wt</i> 3-day time point was set to 1, and the C_N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. <b>C:</b> U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8^∧E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. <b>D and E:</b> U2OS cells were transfected with 2 µg of HPV18 <i>wt</i> minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C_N) was performed. The value obtained from the HPV18 <i>wt</i> 3-day time point was set to 1, and the C_N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 <i>wt</i> replication, whereas panel E shows the effect of E2C-2.</p

Mapping of the HPV18 splice junctions in U2OS cells by 5′ RACE.

<p>The assays were conducted with U2OS cells that had been transiently transfected with the HPV18 genome (timepoints indicated above each gel) and with HPV18-positive U2OS subclone #1.13 cells that were cultured in subconfluent or confluent conditions. (A) 5′ RACE analysis of HPV18 transcripts that were produced in U2OS cells during transient replication. An HPV18-specific primer (Pr3517-1) that binds to a site in the E4 ORF was used. All distinct products (regions marked as 1, 2, 3 and 4) were purified, cloned and sequenced. The natures of the represented transcripts are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116151#s3" target="_blank">results</a> and are shown in Fig. 6. (B) 5′ RACE analysis of RNAs in U2OS cells with an HPV18-specific primer (Pr904-1) that binds to a site in the E7 ORF. The dominant RACE product (size ∼700 bp) represents the spliced transcripts as -233/416- (RNAs A, C and K in Fig. 6). Products representing the RNAs that initiated from promoters P₁₀₂, P₅₂₀ and P₈₁₁ are indicated. (C) 5′ RACE analysis of E1-encoding mRNAs with an HPV18-specific primer (Pr1397-1) that binds to a site in the E1 ORF. The indicated products (1–5) were cloned and sequenced. Products representing the RNAs that initiated from promoters P₁₀₂, P₅₂₀, P₈₁₁ and P₁₁₉₃ are indicated. (D) Primer extension assay of HPV18 genome transiently replicating in U2OS cells. Signals are obtained with HPV18-specific primer Pr881. Lane 1 serves as marker (ΦX174 DNA/HinfI; Promega), lane 2 is HPV18 genome and lane 3 is mock sample. Bands representing transcripts arisen from HPV18 promoters are indicated. (E) Primer extension assay quantitation of transiently replicating HPV18 in U2OS cells. Values indicate proportion of overall promotoral activity.</p

Frequency of human consensus transcription start site motifs in promoter regions.

<p>Frequency of human consensus transcription start site motifs in promoter regions.</p

Transcriptional analyses of HPV18 E2 variants A: Schematic representations of two reporter plasmids.

<p>One reporter consists of full-length native HPV18 URR region acting as a promoter that drives the Firefly luciferase gene. The other reporter uses HPV18 E2-binding sites 3 and 4 as promoter. B: U2OS cells were transfected with the 18URR-Luc+ reporter plasmid and the non-specific pRL-TK reporter and together with different concentrations (0.1 ng, 10 ng, 100 ng and 500 ng) of the HPV18 E2C-1, E2C-2 and E8ˇE2 expression plasmids; 0.1 ng and 100 ng of the HPV18 E2 plasmid were added as controls. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of E2C-1, E2C-2, E8ˇE2 and E2 on the URR promoter activity relative to the reporter alone. Average values of two independent experiments are given. C: U2OS cells were transfected with the 18 E2BS 3&4-Luc+ reporter plasmid and non-specific pRL-TK reporter and together with different concentrations (0.1 ng, 0.5 ng, 1 ng, 3 ng, 5 ng or 10 ng) of HPV18 E2C-2 or 0.1 ng, 1 ng, 10 ng or 100 ng of the BPV1 E2C expression plasmids. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of HPV18 E2C-2 and BPV1 E2C on the E2BS 3&4 promoter activity relative to the reporter alone. Average values of three independent experiments are given. D: U2OS cells were transfected with the 18 E2BS 3&4-Luc+ reporter plasmid and non-specific pRL-TK reporter and together with different concentrations (1 ng, 10 ng and 100 ng) of the HPV18 E2C-1, E8ˇE2 and E2 expression plasmids. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of HPV18 E2C-1, E8ˇE2 and E2 on the E2BS 3&4 promoter activity relative to the reporter alone. Average values of two independent experiments are given. E and F: 5′ RACE analysis of HPV18 transcripts that were produced in U2OS cells during transient replication of <i>wt</i> or different mutant (E2C-2, E2C-1 and 2-E2C) genomes. HPV18-specific primers, Pr904, which binds to a site in the E7 ORF (panel E), and Pr3157 (panel F), which binds to a site in the E4 ORF, were used. Products representing the RNAs that are initiated from promoters P₁₀₂, P₅₂₀ and P₈₁₁ are shown.</p

Summary of the HPV18 transcripts that were mapped in transiently transfected U2OS cells and in subclone #1.13 cells by 5′ RACE analyses with primers Pr1397 (RNA A) and Pr3517-1 (RNAs B-U).

<p>Top, a linear depiction of the HPV18 genome with the ORFs, LCR, E1^∧E4 and E2^∧E8 coding sequences spanned over two exons (dashed line), along with the promoter regions P₁₀₂, P₅₂₀, P₈₁₁, P₁₁₉₃ and P₃₃₈₅ and the major polyadenylation cleavage sites CS 4270 and CS 7298. All transcripts depicted herein (designated with letters A-U, shown at left) are shown with exons (solid boxes) and introns (lines) and with the splicing donor and acceptor sites (nt numbers). The coding potential is described to the right of each transcript. The RNA species previously described in HPV18-infected raft cultures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116151#pone.0116151-Wang1" target="_blank">[18]</a> are indicated by asterisks. In addition, the splicing pattern -3165/3434- (as for RNA H herein) was reported previously in both raft culture and clinical samples (41).</p

Mapping of the HPV18 transcription polyadenylation cleavage sites (CSs) by 3′ RACE.

<p>The assays were conducted in U2OS cells that were transiently transfected with the HPV18 genome (timepoints are indicated above each gel) and in HPV18-positive U2OS subclone #1.13 cells that were cultured in subconfluent or confluent conditions. (A) 3′ RACE analysis of the HPV18 early region CS with the HPV18-specific primer Pr3976. The 3′ RACE product (indicated by an arrow) represents the CS at 4270. (B) 3′ RACE analysis of the HPV 18 late region CS with the HPV18-specific primer Pr7020. Three 3′ RACE products (marked as 1, 2 and 3 and indicated by arrows) represent the CSs at nt 448, nt 7693 and nt 7298, respectively. (C) 3′ RACE analysis with the HPV18-specific primer Pr2500 for analysis of the CSs of E1-encoding transcripts. Three 3′ RACE products (marked as 1, 2 and 3 and indicated by arrows) represent the CSs at nt 4270, nt 3339 and nt 2741, respectively.</p