Biomedical subjects

M Wiskerchen

Publications and source records attributed to M Wiskerchen.

10 recordsLinked to original sources

PubMed1998

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication.

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.

Amino Acid Sequence↗

PubMed1997

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.

Amino Acid Sequence↗

PubMed1996

HIV-1 Nef association with cellular serine kinase correlates with enhanced virion infectivity and efficient proviral DNA synthesis.

We previously reported on the association of Nef with a cellular serine kinase (E.T. Sawal et al., Proc. Natl. Acad. Sci. USA 91, 1539-1543, 1994). In the present study, we further define the Nef sequence requirements for this kinase association and investigate the effect of this kinase association on functions of HIV-1 Nef. We observe that, in addition to the membrane targeting signal and the conserved arg-arg residues within the core region, mutations in the proline-rich domain of Nef also affect its ability to associate with the serine kinase activity. The region encompassing the arg-arg residues of Nef is shown to be important for Nef-mediated cell-surface CD4 down-modulation as well as enhancement of viral growth properties. This is similar to what has previously been observed for the membrane targeting site at the N-terminus of Nef. In contrast, the proline-rich region of Nef is found to be involved in mediating efficient proviral DNA synthesis and the enhanced virion-infectivity function, but is not necessary for CD4 down-modulation by Nef. Thus, it appears that serine kinase association of Nef is necessary for efficient proviral DNA synthesis and for promotion of virion infectivity of Nef viruses, but is dispensable for down-regulation of the CD4 receptor by Nef. These findings define three functional domains of Nef that are required for its interaction with the serine kinase activity and suggest that the cellular interaction events via the myristoylation and arg-arg regions of Nef lie upstream of the interaction event via the proline-rich domain.

CD4 Antigens↗

PubMed1996

Potent human immunodeficiency virus type 1 protease inhibitors that utilize noncoded D-amino acids as P2/P3 ligands.

Noncoded D-amino acids have been designed to replace the quinaldic amide-asparaginyl moiety (P2/P3 ligand) found in several potent human immunodeficiency virus (HIV) protease inhibitors such as LY289612. The substituted nitrogen, optimally an N-methanesulfonyl moiety, served as a CH2CONH2 (asparagine side chain mimic), while the amino acid side chain became the backbone and P3 ligand of these novel inhibitors. Compounds derived from S-aryl-D-cysteine proved to be potent HIV protease inhibitors which also exhibited potent whole cell antiviral activity. Oxidation of the cysteines to the sulfoxide or sulfone oxidation states resulted in significant improvements in potency. For example, the compound derived from N-(methyl-sulfonyl)-2-S-naphthylcysteine sulfone, 17c, was a 3.5 nM inhibitor of HIV protease which inhibited the spread of virus in MT4 cells with an IC50 = 4.3 nM. Compounds 17c,g,i were found to be orally bioavailable in a rat model.

Amino Acids↗

PubMed1995

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.

Base Sequence↗

PubMed1995

Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene.

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity. However, during infection at the nonpermissive temperature of 39.5 degrees C, K136A/E138A is capable of only one round of integration. Mutant virions formed at 39.5 degrees C do not integrate but are indistinguishable from wild-type virions when scored for activity of reverse transcriptase and correct expression and processing of Gag and Pol proteins. We demonstrate that the defect responsible for the ts phenotype of K136A/E138A is localized to a step after proviral formation and integrase protein synthesis but prior to particle maturation. It is the temperature at which the K136A/E138A virion is synthesized, not the temperature at which infection occurs, which determines the ability of the virus to integrate.

Alanine↗

PubMed1992

Baculovirus expression of pestivirus non-structural proteins.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

Animals↗

PubMed1991

Bovine viral diarrhea virus genomic organization.

In previous work, we developed a preliminary description of the genetic organization of the prototypic pestivirus bovine viral diarrhea virus (BVDV). In order to refine this genetic map and to further elucidate the gene products and expression strategy of this virus, we have generated a broad panel of sequence-specific antibody reagents. Use of these reagents not only allowed the identification of several previously undescribed viral polypeptides, but when used in in vivo pulse-chase experiments, they identified precursor polyproteins and processing intermediates. Data generated from these studies provide a more accurate and complete view of viral gene organization, as well as insight into several aspects of protein processing and the gene expression strategy employed by this pestivirus. These experiments also revealed varying stability and turnover rates for the mature BVDV proteins. These latter results have implications for the functional roles of certain gene products.

Animals↗

PubMed1991

Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing.

Bovine viral diarrhea virus (BVDV), the prototypic pestivirus, possesses a positive-strand RNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. We have endeavored to elucidate the mechanisms involved in protein biogenesis by this pestivirus. Here, we present our studies on gene expression from the viral nonstructural protein coding region encompassing the carboxy-terminal 60% of the ORF. Previous sequence and modeling analyses predicted the amino-terminal region of the BVDV nonstructural protein p80 to be a trypsin-like serine proteinase. Using a mammalian cell transient expression system, we show that this region indeed possessed a proteolytic activity and, further, required the serine residue previously predicted to be the putative serine proteinase catalytic site. We found the p80-region proteinase activity was required for proteolytic processing of all viral nonstructural proteins. Cleavage by this activity at the amino and carboxy termini of the p80 protein itself likely occurred intramolecularly (in cis), since we were unable to demonstrate activity in trans at these sites. Cleavages at the three processing sites downstream of the carboxy terminus of p80 were shown to occur in trans. However, p80 proteinase activity alone was not sufficient for cleavage of the last of these sites. Another viral gene product, or specific condition, is implicated as a necessary cofactor for p80 proteinase activity at this site. Pestivirus polyprotein processing can now be compared to similar events by viruses of other groups. Finally, the potential role of p80 proteinase activity in the phenotype of cytopathic biotypes of BVDV is discussed.

Animals↗

PubMed1991

Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity.

The positive-strand RNA genome of pestiviruses contains a single large open reading frame (ORF) extending its entire length and is capable of encoding 450 kDa of protein. Studies have been undertaken with the purpose of elucidating the specific mechanisms involved in the biogenesis of the complete complement of pestivirus proteins. Here, we report on gene expression at the 5' end of the genome of the prototype pestivirus, bovine viral diarrhea virus (BVDV). We demonstrate, using both a cell-free transcription-translation system and a mammalian-cell transient-expression system, that the first protein product of the large ORF of BVDV, the p20 protein, possesses a specific proteolytic activity. The p20 proteinase activity acts to release the p20 protein from the nascent polyprotein. The p20 proteinase activity is not, however, required for downstream glycoprotein processing, indicating translocation of the pestivirus glycoprotein precursor is affected by an internal signal sequence.

Amino Acid Sequence↗