Michael A. Parniak

Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris

Crude extracts of four Chinese herbs, Arctium lappa, Astragalus membranaceus, Andrographis paniculata, and Prunella vulgaris, were assessed in several tissue culture lines for anti-HIV activity and for cytotoxicity. One extract, obtained from P. vulgaris, was able to significantly inhibit HIV-1 replication with relatively low cytotoxicity. The active factor was purified using sequential precipitations with ethanol and n-butanol, followed by reverse-phase and gel permeation high-performance liquid chromatographic separations. The active component was anionic with a molecular weight of approximately 10 kDa. The purified extract inhibited HIV-1 replication in the lymphoid cell line MT-4, in the monocytoid cell line U937, and in peripheral blood mononuclear cells at effective concentrations of 6, 30, and 12.5 micrograms/ml, respectively. Pretreatment of uninfected cells with the extract prior to viral exposure did not prevent HIV-1 infection. By contrast, preincubation of HIV-1 with the purified extract dramatically decreased infectiousness. The purified extract was also able to block cell-to-cell transmission of HIV-1, prevented syncytium formation, and interfered with the ability of both HIV-1 and purified gp120 to bind to CD4. PCR analysis confirmed the absence of HIV-1 proviral DNA in cells exposed to virus in the presence of the extract. These results suggest that the purified extract antagonizes HIV-1 infection of susceptible cells by preventing viral attachment to the CD4 receptor.

In vitro expression analysis of mutations in phenylalanine hydroxylase: Linking genotype to phenotype and structure to function

Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell‐free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site‐directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site (http://www.mcgill.ca/pahdb). Hum Mutat 11:4–17, 1998.

Inhibitors of HIV- I reverse transcriptase

Publisher Summary This chapter discusses the current clinically used reverse transcriptase (RT) inhibitors and some promising new inhibitors still in preclinical development, emphasizing the mechanisms of action of RT inhibitors and the mechanisms of viral resistance that develop upon continued exposure of the virus to these compounds. Once integrated, proviral DNA is therapeutically indistinguishable from cellular DNA and results in persistent HIV infection and the establishment of chronically infected cells that are major contributors to the continued spread of HIV infection in an infected individual. Conversion of HIV genomic RNA into double-stranded viral DNA is a complex process. All chemical steps are catalyzed by the viral enzyme, RT. Because there is no cellular homolog of retroviral RT, the enzyme is an attractive target for the development of HIV therapeutics. Several nucleoside reverse transcriptase inhibitors (Nucleoside RT inhibitors (NRTI)) are in current clinical use. NRTIs are analogs of natural deoxynucleosides, there is the potential that they will be used by normal cellular DNA polymerases, with accompanying toxicity. Thus, the selectivity of NRTI used by HIV-1 RT, compared to that by cellular DNA polymerases, is an important issue in the development of NRTI as therapeutic agents. While many inhibitors of HIV-1 RT DNA polymerase activity have been developed, very few inhibitors of HIV-1 RT ribonuclease H have been described in the chapter. The gene for HIV-1 RT encodes a 66-kDa polypeptide; however, the presumed biologically relevant from of HIV-1 RT is a heterodimer consisting of two subunits of 66 and 51 kDa.

Alterations in protein aggregation and degradation due to mild and severe missense mutations (A104D, R157N) in the human phenylalanine hydroxylase gene (PAH)

Phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine; its activity is the major determinant of phenylalanine disposal. Mutations in the corresponding human gene (PAH), which encodes the human hepatic PAH enzyme, result in hyperphenylalaninemia; the resulting phenotypes can range in severity from mild forms of hyperphenylalaninemia with benign outcome to the severe form, phenylketonuria with impaired cognitive development. This paper describes the detailed characterization of two inherited recessive missense mutations in PAH, c.311C→A (A104D) and [c.470G→A;c.471A→C] (R157N), which are associated, respectively, in the homozygous or functionally hemizygous states, with mild and severe metabolic phenotypes. We used three different in vitro PAH expression systems (in Escherichia coli, cell‐free rabbit reticulocyte lysates, and human embryonal kidney cells), as well as a unique assay for phenylalanine oxidation in vivo. In each system, we observed alterations of PAH function and physical properties, compared with wild‐type enzyme, and differences in relative severity of effects between these two mutations. Pulse‐chase experiments showed increased PAH degradation, probably related to observed aberrations in protein folding and altered oligomerization, as a basic mechanism underlying effects of these missense mutations. Hum Mutat 12:344–354, 1998.© 1998 Wiley‐Liss, Inc.

Incorporation of tRNA into normal and mutant HIV-1

During retroviral assembly, tRNAs are incorporated into the virion, one of which serves as a primer for the reverse transcription reaction. Using two dimensional polyacrylamide gel electrophoresis, we have studied the patterns of tRNAs incorporated into HIV-1 (3B) produced either in the lymphoid cell line H-9 or in the monocytic cell line U937. We have also examined viral tRNA patterns incorporated in a non-infectious, mutant virion which lacks pol gene products and processed gag protein. Our results lead to the following conclusions: 1) tRNA incorporated into HIV-1 is a select subpopulation of the host-cells tRNA. 2) The type of tRNA incorporated into the virion is dependent upon cell type. 3) There can be multiple species of tRNA of similar mobilities tightly associated to the viral genome. 4) The packaging of putative primer tRNA into virions requires either the synthesis of pol gene products, the processing of gag proteins, or both, while the incorporation of non-primer tRNAs does not.