Lester L. Gutshall

Resistance profile of a hepatitis C virus RNA-dependent RNA polymerase benzothiadiazine inhibitor.

ABSTRACT Recently, a benzo-1,2,4-thiadiazine antiviral agent (C21H21N3O4S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 μM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication

ABSTRACT Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of theFlaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 μM, all of the mutations except one had Kms that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32°C, 37°C, or 39.5°C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.

His-tag binding by antibody C706 mimics β-amyloid recognition

Alzheimers disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ42 peptide leading to plaque formation is believed to play a central role in Alzheimers disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P21 and C2. Although the Fab was crystallized in the presence of Aβ16, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright