Human monoclonal antibody MBL-HCV1 delays HCV viral rebound following liver transplantation: a randomized controlled study.

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-α and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated and reduced viral load for a period ranging from 7 to 28 days. Median change in viral load (log10 IU/mL) from baseline was significantly greater (p=0.02) for the antibody-treated group (range -3.07 to -3.34) compared to placebo group (range -0.331 to -1.01) on days 3 through 6 posttransplant. MBL-HCV1 treatment significantly delayed median time to viral rebound compared to placebo treatment (18.7 days vs. 2.4 days, p<0.001). As with other HCV monotherapies, antibody-treated subjects had resistance-associated variants at the time of viral rebound. A combination study of MBL-HCV1 with a direct-acting antiviral is underway.

Mammalian reovirus L2 gene and lambda2 core spike protein sequences and whole-genome comparisons of reoviruses type 1 Lang, type 2 Jones, and type 3 Dearing.

The reovirus L2 genome segment encodes the core spike protein lambda2, which mediates enzymatic reactions in 5' capping of the viral plus-strand transcripts. Complete nucleotide-sequence determinations were made for the L2 genome segments of eight mammalian reoviruses, including the prototype isolates of serotypes 1 and 2: Lang (T1L) and Jones (T2J), respectively. Each L2 segment was found to be 3912 or 3915 bases in length. Partial nucleotide-sequence determinations were also made for the 3916-base L2 segment of reovirus type 3 Dearing (T3D), the prototype isolate of serotype 3. The whole-genome sequence of reovirus T3D was reported previously. The T1L L2 analysis represents completion of the whole-genome sequence of that isolate as well. The T2J L2 analysis leaves only the sequence of the M1 segment yet to be reported from the genome of that isolate. The T2J M1 sequence made available from analysis in another lab was used for initiating whole-genome comparisons of reoviruses T1L, T2J, and T3D in this report. The nine L2 gene sequences and deduced lambda2 protein sequences were used to gain further insights into the biological variability, structure, and functions of lambda2 through comparisons of the sequences and reference to the crystal structure of core-bound lambda2. Phylogenetic comparisons suggest the presence of three evolutionary lines of divergent L2 alleles among the nine isolates. Localized regions of conserved amino acids in the lambda2 crystal structure include active-site clefts of the RNA capping enzyme domains, sites of interactions between lambda2 domains within the pentameric spike structure, and sites of interaction between lambda2 subunits and other proteins in viral particles.

Reovirus nonstructural protein muNS binds to core particles but does not inhibit their transcription and capping activities.

Previous studies provided evidence that nonstructural protein muNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455-466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of muNS. Given the possible role of muNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant muNS can bind to cores in vitro. The muNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of muNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of muNS were bound. Thus, the muNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the muNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to muNS. The muNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing muNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins mu1 and sigma3. These findings are consistent with the presence of muNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, muNS may block or delay outer-capsid assembly and allow continued transcription by these particles.

Mammalian reovirus M3 gene sequences and conservation of coiled-coil motifs near the carboxyl terminus of the microNS protein.

Nucleotide sequences of the mammalian orthoreovirus (reovirus) type 1 Lang and type 2 Jones M3 gene segments were newly determined. The nucleotide sequence of the reovirus type 3 Dearing M3 segment also was determined to compare with a previously reported M3 sequence for that isolate. Comparisons showed Lang and Dearing M3 to be more closely related than either was to Jones M3, consistent with previous findings for other reovirus gene segments. The microNS protein sequences deduced from each M3 segment were shown to be related in a similar pattern as the respective nucleotide sequences and to contain several regions of greater or less than average variability among the three isolates. Identification of conserved methionine codons near the 5' ends of the Lang, Jones, and Dearing M3 plus strands lent support to the hypothesis that microNSC, a smaller protein also encoded by M3, arises by translation initiation from a downstream methionine codon within the same open reading frame as microNS. Other analyses of the deduced protein sequences indicated that regions within the carboxyl-terminal third of microNS and microNSC from each isolate have a propensity to form alpha-helical coiled coils, most likely coiled-coil dimers. The new sequences will augment further studies on microNS and microNSC structure and function.

Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the lambda1 protein.

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein.