Drugs as molecular tools.
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Antiviral Agents↗
Biomedical subjects
D C Pevear
Publications and source records attributed to D C Pevear.
Treatment of the picornavirus common cold by inhibitors of viral uncoating and attachment.
The human rhinoviruses are the leading cause of the ubiquitous, mild, and self-limiting infections generally referred to as the common cold. Considerable research effort has been expended in the search for well tolerated antiviral agents capable of preventing and treating the common cold. Although no antirhinovirus drug is yet commercially available, considerable progress has been made in the discovery and development of novel, viral specific inhibitors of rhinovirus replication. This report reviews the history and current status of the research that has focused on inhibitors of the early steps in the virus life cycle: attachment to the cellular receptor and uncoating of the viral RNA. Molecules directed at these targets currently possess the greatest potential for generating a safe and efficacious treatment for the rhinovirus common cold.
Antiviral Agents↗
A model for compounds active against human rhinovirus-14 based on X-ray crystallography data.
A number of (oxazolinylphenyl)isoxazoles have been synthesized and tested against human rhinovirus-14 (HRV-14). Several of the more active compounds have been examined by X-ray crystallography and their orientation in the compound binding site on the capsid protein of HRV-14 has been determined. Based on the minimum inhibitory concentration against HRV-14 and the X-ray conformation of the compounds, a model has been developed which distinguishes between the space-filling properties of the active and inactive compounds in this series. The model was generated by overlaying composite structures and comparing the van der Waals generated volume maps. The results of this study indicate that inactive compounds display areas of excessive bulk particularly around the phenyl ring, while the active compounds occupy space below the pore area of the compound binding site.
Antiviral Agents↗
Localization of genomic regions specific for the attenuated, mouse-adapted poliovirus type 2 strain W-2.
In order to begin to elucidate the genomic basis of the attenuation of mouse-adapted, poliovirus type 2 strain W-2 (PV2/W-2), we have cloned and sequenced the virus and compared it with the virulent, mouse-adapted PV2/Lansing strain. In addition, we have performed computer-generated comparisons of PV2/W-2 to the non-mouse-adapted, attenuated PV2/Sabin strain to determine whether mutational patterns occur that result in attenuation. The PV2/W-2 genome is 7434 nucleotides in length, which is three bases shorter than PV2/Lansing. The 5' non-coding region of PV2/W-2 is 747 nucleotides in length (compared to 744 in PV2/Lansing) and shares 98.8% identity with PV2/Lansing and 82.3% identity with PV2/Sabin. Overall, the PV2/W-2 polyprotein (2205 amino acids) is two amino acids shorter than that of either PV2/Lansing or PV2/Sabin (2207 amino acids). These contiguous deletions fall within the P3-D region (polymerase). Within these 2205 amino acid residues only 26 differences were observed between PV2/W-2 and PV2/Lansing (98.8% identity), whereas 92 occurred between PV2/W-2 and PV2/Sabin (95.8% identity). The 3' non-coding region of PV2/W-2 is 72 nucleotides in length and shares 100% identity with PV2/Lansing and 98.6% identity with PV2/Sabin. Amino acid changes in the capsid protein region occurred in neutralization sites 1 and 3, areas previously shown to be important for pathogenicity. The cleavage site between non-structural proteins P2-C/P3-A consisted of a glutamine-serine pair, in contrast to other sequenced polioviruses which have a glutamine-glycine dipeptide.
Amino Acid Sequence↗
Crystal structure of human rhinovirus serotype 1A (HRV1A).
The structure of human rhinovirus 1A (HRV1A) has been determined to 3.2 A resolution using phase refinement and extension by symmetry averaging starting with phases at 5 A resolution calculated from the known human rhinovirus 14 (HRV14) structure. The polypeptide backbone structures of HRV1A and HRV14 are similar, but the exposed surfaces are rather different. Differential charge distribution of amino acid residues in the "canyon", the putative receptor binding site, provides a possible explanation for the difference in minor versus major receptor group specificities, represented by HRV1A and HRV14, respectively. The hydrophobic pocket in VP1, into which antiviral compounds bind, is in an "open" conformation similar to that observed in drug-bound HRV14. Drug binding in HRV1A does not induce extensive conformational changes, in contrast to the case of HRV14.
Amino Acid Sequence↗
Inhibitors of viral uncoating.
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Antiviral Agents↗
Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors.
A series of eight antiviral compounds complexed with human rhinovirus 14 (HRV-14) were previously shown to displace segments of polypeptide chains in the floor of the "canyon" by as much as 0.45 nm in C-alpha positions from the native conformation (J. Badger, I. Minor, M. J. Kremer, M. A. Oliveira, T. J. Smith, J. P. Griffith, D. M. A. Guerin, S. Krishnaswamy, M. Luo, M. G. Rossman, M. A. McKinlay, G. D. Diana, F. J. Dutko, M. Fancher, R. R. Rueckert, and B. A. Heinz, Proc. Natl. Acad. Sci. USA 85:3304-3308, 1988). Because the canyon is thought to serve as the viral receptor-binding site (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H. J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London] 317:145-153, 1985; M. G. Rossmann and R. R. Rueckert, Microbiol. Sci. 4:206-214, 1987), these compounds were assessed for their ability to block adsorption of HRV-14 to HeLa cell membrane receptors. In parallel experiments, the compounds were assessed directly for antiviral activity in an in vitro plaque reduction assay in intact HeLa cells. All eight compounds blocked the adsorption of 50% of HRV-14 at approximately the same concentration required to reduce the number of visible plaques by 50% (MIC). A structurally related compound which was inactive in the plaque reduction assay had no effect on HRV-14 binding. A drug-resistant mutant of HRV-14 (Leu-1188), which was less sensitive to the eight compounds in plaque reduction assays was similarly less sensitive in the adsorption assay. We propose that the conformational changes in the floor of the HRV-14 canyon induced by these compounds substantially decrease adsorption of the virion to its receptor. These results provide further evidence for the role of the HRV canyon in receptor binding.
Adsorption↗
Insights into Theiler's virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains.
Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which can be divided into two subgroups based primarily on their neurovirulence after intracerebral inoculation: the highly virulent GDVII group and the less virulent TO strains. To begin to elucidate the molecular basis of neurovirulence of the two TMEV subgroups, we have cloned and sequenced the entire 8105 nucleotide RNA genome of the highly virulent GDVII virus and compared it to the less virulent BeAn 8386 virus (D. C. Pevear, M. Calenoff, E. Rozhon, and H. L. Lipton (1987) J. Virol. 61, 1507-1516). The viruses are 90.4% identical at the nucleotide level. The highest level of nucleotide identity is in the 5' and 3' noncoding regions of the RNAs (95.5 and 99.2%, respectively): regions believed to be important for control of viral RNA synthesis, initiation of translation, encapsidation, and virion uncoating. The 2303 amino acid polyproteins of BeAn and GDVII viruses are 95.7% identical at the amino acid level (99 of 2303 residues differed). Thirty-nine of these amino acid differences occur in the three surface coat proteins, VP1 (20 differences), VP2 (10 differences), and VP3 (9 differences), while the remainder of the changes are distributed throughout the polyprotein. Although these levels of identity are too low to determine where neurovirulence maps based solely on nucleotide sequence analysis, having the complete sequence will facilitate construction of recombinant BeAn-GDVII viruses to be used for this purpose.
Amino Acid Sequence↗
Three-dimensional model of the capsid proteins of two biologically different Theiler virus strains: clustering of amino acid difference identifies possible locations of immunogenic sites on the virion.
To explore structural features of the Theiler murine encephalomyelitis virion, we have constructed a three-dimensional model of the capsid proteins (VP1, VP2, and VP3) of the BeAn strain based on the atomic coordinates of the closely related Mengo virus. By superimposition of amino acid differences between BeAn virus and another Theiler virus strain, GDVII, on the three-dimensional model, clusters of differences were found in four distinct sites; the VP1 third corner, the VP2 "puff," and the VP3 first corner and "knob." These clusters, which are found on the surface of the virion, may represent neutralizing immunogenic sites that have come under selective pressure from neutralizing antibodies. Furthermore, the putative viral receptor binding site ("pit") of the two Theiler virus strains was found to be markedly conserved.
Amino Acid Sequence↗
Sequence comparison of a highly virulent and a less virulent strain of Theiler's virus. Amino acid differences on a three-dimensional model identify the location of possible immunogenic sites.
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Amino Acid Sequence↗
Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins.
Following intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized by mononuclear cell-rich infiltrates in the central nervous system. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility class II-restricted delayed-type hypersensitivity, which temporally correlates with disease onset and remains chronically elevated in susceptible mice. This study examined the fine specificity of class II-restricted T cell responses in TMEV-infected mice to better define the relevant virus-encoded T cell determinant(s) responsible for triggering the demyelinating process, and to determine if class II-restricted neuroantigen-specific autoimmune responses could be detected in mice with TMEV-induced demyelination. The data clearly show that T cell responses in TMEV-infected mice are directed against determinants shared by closely related TMEV strains and are cross-reactive with related picornaviruses, such as encephalomyocarditis virus. In contrast, class II-restricted autoimmune responses against syngeneic mouse spinal cord homogenate and the two major protein components of myelin, myelin basic protein and proteolipid protein, are not demonstrable in susceptible SJL/J mice undergoing chronic TMEV-induced demyelinating disease, but are readily seen in SJL/J mice displaying chronic, relapsing experimental allergic encephalomyelitis. Cross-reactivity (or lack thereof), as determined by functional T cell analyses, was found to correlate with the extent of exact amino acid homology between the TMEV capsid proteins, the two neuroantigens, and related picornaviruses. The data thus do not support a major role for autoimmune responses against myelin proteins in TMEV-induced demyelinating disease, but are consistent with our previously proposed hypothesis that TMEV-specific T cell responses constitute a major effector mechanism of myelin breakdown.
Animals↗
Characterization and specificity of humoral immune responses to Theiler's murine encephalomyelitis virus capsid proteins.
Humoral antibody responses to Theiler's murine encephalomyelitis virus (TMEV) capsid proteins were examined. Rabbit antisera produced against the native BeAn strain of TMEV and against the isolated capsid proteins (VP1, VP2 and VP3) were tested for their ability to bind or neutralize virus and to inhibit the virus-induced haemagglutination of human O+ erythrocytes. Western immunoblotting analysis showed that isolated VP1, VP2 and VP3 each primed for a specific antibody response, but that native virions primed for antibodies specific for VP1 and VP2, but not VP3. Virus neutralization studies revealed that a dominant TMEV neutralizing determinant(s) lay on VP1, as did the haemagglutinating determinant. The possible location of the neutralizing epitopes are discussed on the basis of molecular modelling of the predicted amino acid sequence of TMEV from that of the closely related Mengo virus for which the three-dimensional structure is known.
Animals↗
Lack of correlation between serum titres of interferon alpha, beta, natural killer cell activity and clinical susceptibility in mice infected with two isolates of lymphocytic choriomeningitis virus.
Intracerebral infection of adult immunocompetent mice with most strains of lymphocytic choriomeningitis virus (LCMV) caused a systemic infection and led to severe meningoencephalitis and death due to the induced T cell immune response. The susceptibility of congenic mice to the two plaque variants Docile and Aggressive of LCMV strain UBC was shown to be mouse strain-dependent. To investigate the possible correlation between acid-stable interferon (IFN) and natural killer (NK) cell responses and the susceptibility to the two UBC LCMV substrains, serum titres of acid-stable antiviral activity, presumably IFN-alpha, beta and NK cell activities were determined in various mouse strains at different times after intracerebral infection. The two viral isolates induced comparable IFN-alpha, beta serum titres and caused similar NK activities in the same mouse strain. Between different mouse strains, marked differences in the kinetics and amount of IFN production were observed, yet there was no correlation with the susceptibility to the two UBC LCMV substrains. Additionally, there was no correlation between the magnitude of the IFN-alpha, beta serum titres and the NK activities induced in the spleen by the viral inocula. Overall, the findings suggest that levels of circulating IFN-alpha, beta are only of minor importance for the development of LCM disease.
Animals↗
Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses.
Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which constitute a separate serological group within the picornavirus family. Persistent TMEV infection in mice provides a relevant experimental animal model for the human demyelinating disease multiple sclerosis. To provide information about the TMEV classification, genome organization, and protein processing map, we determined the complete nucleotide sequence of the TMEV genome and deduced the amino acid sequence of the polyprotein coding region. The RNA genome, which is typical of the picornavirus family, is 8,098 nucleotides long. The 5' untranslated region is 1,064 nucleotides long (making it the longest in the picornavirus family after the aphthoviruses) and lacks a poly(C) tract. Computer-generated comparison of the 5' and 3' noncoding regions and polyprotein revealed the highest level of nucleotide and predicted amino acid identity between the TMEV and the cardioviruses encephalomyocarditis virus (EMCV) and Mengo virus. The TMEV polyprotein, which appears to be processed like EMCV since the amino acids flanking the putative proteolytic cleavage sites have been conserved, begins with a short leader peptide followed by 11 other gene products in the standard L-4-3-4 picornavirus arrangement. Because of these similarities, we propose that the TMEV be grouped with the cardioviruses. However, since TMEV and EMCV have different biophysical properties and show no cross-neutralization, they most likely belong in a separate cardiovirus subgroup.
Amino Acid Sequence↗
Severe and transient pancytopenia associated with a chronic arenavirus infection.
A so-called 'docile' strain of Lymphocytic Choriomeningitis Virus (LCMV) lacks the ability to cause the fatal central nervous system syndrome, commonly associated with most other strains of this virus, in C3HeB/FeJ mice. Hematological evaluation during a 5 week period revealed that every mouse experienced a pancytopenia which was the most severe around three weeks post-infection. The abnormal red blood cell (RBC) morphology seen in the peripheral blood along with the increased reticulocyte count and marked erythroid hyperplasia in the bone marrow indicated peripheral destruction, rather than stem cell inhibition, as the mechanism causing the anemia. An increase in the 59Fe uptake into the blood confirmed the fact that there was no loss in the erythropoietic capabilities in these mice at this time. Although it was clear that the RBCs were being destroyed in the periphery, there was no evidence of a microangiopathic hemolytic anemia nor of a direct viral infection of these cells. Cyclophosphamide treatment, however, prevented the phenomenon. Thus, it seemed likely that the virus-induced hemolytic anemia in these mice was immune-mediated. The late, but not the early drop in the white blood cell counts and the thrombocytopenia, on the other hand, could be traced to granulocyte and megakaryocyte inhibition in the bone marrow.
Anemia, Hemolytic, Autoimmune↗
Lymphocytic choriomeningitis virus-induced disease of the central nervous system and the "antigen-sink" hypothesis.
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Animals↗
Lack of correlation between cytotoxic T lymphocytes and lethal murine lymphocytic choriomeningitis.
Adoptive transfer of lymph node and spleen cells from mice infected with LCM virus to similarly infected immunocompromised recipients has been the classic way to demonstrate the lethal role of T cells in the CNS disease caused by this virus. Isolation and adoptive transfer techniques are presented here which show that Thy-1+ cells isolated from the meningeal infiltrates (MI) of LCM virus-infected mice possess this property. We compared various T cell functions of MI cells taken from mice infected with two strains of LCM virus differing markedly in their pathogenicities. One of these strains, termed aggressive, caused a typical, invariably fatal, CNS disease within 7 to 10 days after infection. The other virus, termed docile, killed few mice after the standard intracerebral inoculation, and could persist in the mice for 6 mo or more. The yields of MI leukocytes from mice infected with docile virus varied from 50 to 100% of those found in mice infected with aggressive virus (3 X 10(6) cells/brain). On a cell-to-cell basis, the CTL activity in the MI of mice infected with docile virus ranged from 50 to 100% of that found in the MI of mice infected with aggressive virus. MI cells from mice infected with aggressive virus consistently caused lethal disease by adoptive transfer into immunocompromised (irradiated) recipients infected with either strain of virus. All attempts to induce lethal disease by adoptive transfer of MI cells (or splenocytes) from mice infected with docile virus into irradiated recipients failed. The latter experiments with the docile-MI cells were performed with six times the number of aggressive-MI cells needed to kill irradiated recipients by adoptive transfer. The possible reasons for this discordance between CTL and in vivo killer function are discussed.
Animals↗
Immune recognition of tumor cells in mice infected with Pichinde virus.
Pichinde virus (PV), a member of the Arenaviridae family, protects mice from a lethal inoculation with the sarcoma 180 (S180) tumor cell line. Virus replication, which is required for protection, occurs primarily in the spleen and tumor. During the first 4 days, elevated natural killer (NK) cell activity parallels an increase in serum interferon in PV-infected mice. On day 7 after infection virus-specific cytotoxic T cells (CTLs) are found in the mouse. This strong response peaks on day 13 and gradually declines over the next 17 days. The tumor-specific CTL response appears more slowly and is less intense than the virus-specific response, especially in the uninfected mouse. However, CTLs from either type of mouse recognize PV-infected tissue culture S180 target cells better than uninfected ones. Even though the primary tumor-specific immune response appears weak, mice that have cleared both virus and tumor are refractory to a subsequent challenge with S180 cells and rapidly produce tumor-specific CTLs. Thus, our data indicate a number of ways in which virus infection could lead to immune elimination of tumors: (1) Virus-induced interferon stimulates NK-cell activity, which in turn could control tumor load until a specific response is mounted against the S180 cells; (2) early onset of the tumor-specific T-cell response could be brought about by viral-enhanced tumor antigen presentation to the immune system; and (3) the tumor-specific T-cell response could be augmented through a "bystander' phenomenon involving factors associated with T cells responding specifically and vigorously to the virus itself.
Animals↗