An in vitro Flaviviridae replicase system capable of authentic RNA replication.

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.

Size and conformational stability of the hepatitis A virus used to prepare VAQTA, a highly purified inactivated vaccine.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors.

We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.

Peptide substrate cleavage specificity of the human cytomegalovirus protease.

The human cytomegalovirus UL80 gene encodes an 80-kDa precursor polyprotein whose N-terminal 256-amino acid domain is a protease. This enzyme cleaves a specific peptide bond that results in its own release from the precursor, as well as a peptide bond near the C terminus of the viral assembly protein. The latter cleavage is apparently required for encapsidation of the viral genomic DNA and maturation of the viral capsid. A series of peptide substrates, representing the assembly protein cleavage site, was used to study the enzyme's substrate requirements and specificity. It was found that efficient cleavage minimally required the amino acid residues spanning the P4 to P4' positions. Substitution at any of these residues adversely affected the reaction. Conservation of the hydrophobic residues at P3 and P4 was essential. In addition, cleavage of a peptide representing the protease domain release site was reduced almost 100-fold relative to cleavage of the assembly protein maturation site peptide substrate.

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles.

To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full-length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L-687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR.

Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.

We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Visualization of bacterial polysaccharides by scanning transmission electron microscopy.

Highly purified capsular polysaccharides of Neisseria meningitidis groups A, B, and C have been visualized by high resolution Scanning Transmission Electron Microscopy (STEM). Spheroidal macromolecules approximately 200 A in diameter are characteristic of the Meningococcus A and C polysaccharides whereas filaments that are 400-600 A in length are found in Meningococcus B polysaccharide preparations. Filaments are occasionally found associated with the spheroidal Meningococcus A and C polysaccharides and it is proposed that these structures are composed of a long (1-4 microns) filament or filaments that are arranged in spheroidal molecules or micelles of high molecular weight. The Meningococcus B polysaccharide, by contrast, is a short flexuous filament or strand of relatively low molecular weight. A relationship between morphology and antigenicity is proposed.

Viral etiology of age-dependent polioencephalomyelitis in C58 mice.

The etiology of immune polioencephalomyelitis (IPE) and the mechanisms of resistance to IPE induction were investigated in C58 mice. IPE was found to be induced by a lipid-solvent-sensitive, filterable replicating agent present in line Ib leukemic cell suspensions. IPE was serially transmitted in immunosuppressed mice with filtered extracts of spleens from diseased animals. The IPE-inducing activity of Ib cell extracts was abolished by chloroform or deoxycholate. Gel filtration of Ib cell extracts showed that the IPE agent has a molecular weight of at least 10(7). Electron microscopy of the active fractions from columns and of spinal cord extracts from mice with IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE revealed a virus-like particle, 40 nm in diameter, which is probably the IPE agent. Administration of cyclophosphamide at various times after challenge increased the incidence of IPE in mice, suggesting that IPE is not autoimmune mediated. Immunosuppression resulted in maintenance of high levels of IPE agent in the central nervous system tissue, while immunization resulted in low levels. Moreover, immunized mice produced neutralizing antibodies. These data suggest that antibodies help restrict the amount of IPE agent in the nervous tissue, and that this restriction is required for resistance to IPE induction in C58 mice.

Infectious hepatitis (hepatitis A) research in nonhuman primates.

A satisfactory animal model has been found for laboratory studies of human hepatitis A-namely, the white-moustached marmoset (Saguinus mystax). With this species it has been possible to perform serum-neutralization tests and to develop immune-adherence and complement-fixation tests demonstrating antigen and antibody to the virus. The recent work in marmosets has also led to determination of the agent's characteristics: it most closely resembles the enteroviruses of the picornavirus family. These advances open the way for development of a routine serologic test for diagnosis of the disease, of a human immune globulin for general use that would be precisely standardized for hepatitis A antibody, and, ultimately, of a vaccine. They also provide bases for epidemiologic studies that could reveal nonspecific measures for the disease's control. In addition, there is indication that marmosets could be used for safety control of the hepatitis B vaccine that has already been developed. An adequate supply of S. mystax-threatened by recent embargoes on their exportation-is essential to continuation of this work. The question of marmoset supply, both in the short term and over the long range, deserves serious review.

Biophysical and biochemical properties of CR326 human hepatitis A virus.

CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 mmu spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests by infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, identical 27 mmu viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The buoyant density of the virus in CsCl was 1.34 and it was heat (60 C), ether, and acid stable but was destroyed by heat (100 C), formalin (1:4000), and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.

Physical, chemical and morphologic dimensions of human hepatitis A virus strain CR326 (38578).

CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 nm spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests for infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, indentical 27 nm viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The bouyant density of the virus in CsCl was 1.34 and it was heat (60 degrees), ether and acid stable but was destroyed by heat (100 degrees), formalin (1:4000) and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.