Kinetic comparisons of amniotic fluid inactive renin and renal renin using synthetic and human renin substrates

Inactive renin has been isolated from pooled amniotic fluid and purified ~642-fold. Prior to activation the isolates has ~4% of the activity found after activation. The observation is similar to that reported for inactive renin from chorionic cell culture and suggests a placental origin of amniotic fluid inactive renin.Using plasma from an estrogen-treated woman, renin substrate was recovered free of renin and inactive renin and a portion was separated into NMW and HMW components. The NMW form constituted ~93% and the HMW form ~7% of the renin substrate.Amniotic fluid inactive renin was used for determinations of enzyme-substrate kinetics with the pooled, NMW, and HMW plasma substrate and tetradecapeptide synthetic substrate, and the results were compared to similar determinations using standard renal renin. Using synthetic substrate, the kinetics of renal renin and amniotic fluid inactive renin before and after activation were similar. The kinetics of renal renin with pooled, NMW, and HMW plasma substrate were also similar.Amniotic fluid inactive renin had a lower Km with pooled than with NMW substrate, however, which resulted from a significantly smaller Km with HMW component. Although the affinity constants with pooled substrate were not different for renin and inactive renin, the Km of inactive renin was significantly less with the HMW component of plasma renin substrate.The observation are compatible with a role for placental inactive renin in normal pregnancy and suggest the possibility of a further role in hypertensive pregnancy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25955/1/0000021.pd

A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5′ nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/IIIHC) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/IIIHC genome (within the 3′NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5′NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5′NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5′NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3′NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/IIIHC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5′NTR, 3′NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses

Parenchymal Expression of CD86/B7.2 Contributes to Hepatitis C Virus-Related Liver Injury

Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single- and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4(+) T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4(+) T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection

Replication activities of chimeric GBV-B/HCV replicon RNAs.

<p>(A) G418-resistant colony forming activities of the indicated chimeric replicons (black bars, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g001" target="_blank">Fig. 1</a>) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10⁶ cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of parental GBV-B replicon (grey bar) set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 3×10⁴ transfected cells were plated are shown above the graph. (B) Total RNA was extracted from 2–3 cell clones individually picked and expanded after transfection with chimeric replicon GBneoD/III^HC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g001" target="_blank">Fig. 1</a>) or parental GBV-B replicon neo-RepD and analyzed by Northern blot with a riboprobe specific for GBV-B positive-strand RNA after electrophoresis on a denaturing agarose gel. As controls, RNA from mock-transfected cells (mock), as well as various quantities of neo-RepD synthetic RNA transcripts (10⁶, 10⁷, 10⁸ genome equivalents) mixed with cellular RNA from mock-transfected cells, were loaded on the same gel and processed in parallel. Viral RNA and housekeeping β-actin mRNA, detected by a specific riboprobe as a loading control, are indicated by filled and open arrowheads, respectively. Dotted lines indicate where noncontiguous lanes that belong to the same Northern blot image have been brought together.</p

Translational activities of 3′NTR mutant replicons in cB76.1/Huh7 cells

a<p>Replicon RNAs containing 3′NTR mutations or deletions correspond to those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g005" target="_blank">Fig. 5A</a>, but encode firefly luciferase (F-Luc) in place of neomycine phosphotransferase. Luc-RepDΔIII and LucD-GAA control RNAs are described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g002" target="_blank">Fig. 2</a>.</p>b<p>F-Luc RLU activities were determined 4 hours after transfection of equal amounts (5 µg) of replicon RNAs into cB76.1/Huh7 cells, and expressed relatively to Luc-RepD set at 100% after normalization with respect to total protein quantities assayed (mean±SD from two independent transfections, each monitored in duplicate wells).</p

Translational activities of chimeric GBV-B/HCV replicon RNAs.

<p>(A) Chimeric replicon RNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g001" target="_blank">Fig. 1</a>) and parental GBV-B replicon RNA neo-RepD were translated <i>in vitro</i> in rabbit reticulocyte lysates. A representative PAGE-SDS pattern of the first cistron products, i.e. fusion polypeptides between N-terminal core residues and neomycine phosphotransferase (ΔC-neo, open arrowhead), and of second cistron cleavage products for normalization purposes (GBV-B NS3, filled arrowhead), is shown. Note that ΔC-neo polypeptides have different electrophoretic mobilities depending on the number and nature of N-terminal core residues fused to neo (see text). Translational activities of chimeric replicon RNAs are represented as mean±SD relative PhosphorImager volumes for ΔC-neo products quantified from quadruplicate PAGE-SDS gels and normalized with respect to corresponding NS3 PhosphorImager volumes, expressed relatively to neo-RepD volumes set at 100% (Table). (B) A scheme of the reporter replicons used to monitor translational activities in cells is shown at the top of the panel. The product encoded by the first cistron of this replicon is a fusion protein between N-terminal core residues and firefly luciferase (F-Luc). Control reporter RNAs include a translation-deficient GBV-B F-Luc RNA lacking 5′NTR domain III (Luc-RepDΔIII) and a replication-deficient GBV-B F-Luc RNA with a mutation in the RNA polymerase active site (LucD-GAA). Equal amounts (5 µg) of replicon RNAs were transfected into cB76.1/Huh7 cells. Relative F-Luc activities determined at 4 h post-transfection were expressed after normalization with respect to total protein quantities assayed and relatively to values obtained for parental Luc-RepD, set at 100%. Shown are means±SD of four independent transfections, each monitored in duplicate wells.</p

Summary of the infectivity of mutated chimeric GB/III^HC genomes <i>in vivo</i> and sequencing of corresponding viruses

a<p>Combinations of nucleotide substitutions introduced into chimeric genome GB/III^HC in the indicated genomic regions (—: no substitution). Numbering refers to nucleotide position within genome-length GBV-B RNA. Substitutions U₂₃₇₆C and U₇₁₅₂C lead to amino acid changes Ile₆₄₄Thr and Val₂₂₃₆Ala, respectively.</p>b<p>The infectivity of mutated chimeric RNAs was assessed by intrahepatic inoculation of corresponding synthetic RNAs into 1 or 2 tamarins and was scored “+” when viremia developed according to wild-type-like profile and peak viremia was ≥3.5×10⁷ ge/mL, “+/−” when viremia was sporadic and ≤2.5×10⁴ ge/mL, and “−” when viremia remained negative or ≤1.5×10³ ge/mL at 1–2 scattered time points.</p>c<p>Mutations additionally found in the corresponding chimeric viruses rescued <i>in vivo</i> (numbering in the 5′NTR refers to nucleotide position within the chimeric GBV-B/HCV 5′NTR). Substitutions G₅₂₂₅A and C₅₂₂₆U lead to amino acid changes Ala₁₅₉₄Thr and Ala₁₅₉₄Val, respectively. —: no mutation was found in the sequenced regions. NA: not applicable, either because there was no virus rescued or because the viral titers were too low to proceed to sequencing.</p

Replication activities of replicons with mutated 3′NTR.

<p>(A) The chimeric or mutated nature of the 5′ and 3′ NTRs of the indicated chimeras is depicted on the left and right of the figure, respectively, below the GBV-B subgenomic replicon scheme (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004419#pone-0004419-g001" target="_blank">Fig. 1</a>). In the 5′NTRs, white and black boxes correspond to GBV-B and HCV sequences, respectively. Within the 3′NTR of GBV-B, dark grey boxes correspond to the poly(U) tract, stars to the indicated nucleotide substitutions (numbering refers to nucleotide positions within GBV-B genome-length cDNA), and broken lines to the extent of the nucleotide deletion. Positions of translation initiator (AUG) and termination (UGA) codons are indicated by arrows. (B) G418-resistant colony forming activities of the indicated RNAs with a GBV-B 5′NTR (grey bars) or a chimeric 5′NTR containing HCV domain III (black bars) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10⁶ cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of neo-RepD set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 5×10⁵ transfected cells were plated are shown above the graph for each replicon.</p