Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization.

Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.

Cleavage of recombinant and cell derived human immunodeficiency virus 1 (HIV-1) Nef protein by HIV-1 protease.

Recombinant purified Nef protein of HIV-1, as well as Nef protein derived from extracts of permanently HIV-1 infected glioblastoma cells and monocytes, are specifically cleaved by the HIV-1 protease. Nef cleavage products in cellular extracts treated with protease showed identical molecular weights as those obtained by digestion of purified Nef with recombinant HIV-1 protease. Since cellular extracts were prepared by detergent and mechanical lysis it cannot be excluded that physiological cytoplasmic conditions were altered. The lack of Nef cleavage by endogenous HIV-1 protease in infected cells might be due to low concentrations of viral protease and the presence of Gag precursor molecules as natural substrate. Using a panel of monoclonal antibodies two cleavage fragments of 19 kDa and 8 kDa were defined. The cleavage site was located by microsequencing between amino acid 57 and 58 (AW*LEAQEEEEVGF). The conserved cleavage motif within HIV-1 Nef suggests a potential biological function of Nef processing.

Detection and characterization of T-cell leukemia virus-like proviral sequences in PBL and tissues of baboons by PCR.

The "high lymphoma-prone" baboon stock (Papio hamadryas) of the Sukhumi Primate Center colony is characterized by a high prevalence of antibodies to the STLV-I/HTLV-I type of retrovirus and a high manifestation of human ATL-type (adult T-cell leukemia/lymphoma) malignancies (Yakovleva et al., this symposium). This is in contrast to other primate colonies and wild monkeys, which have low seroprevalence and very few if any ATL-type T-cell malignancies. To characterize the type of T-cell lymphoma retrovirus involved in the Sukhumi disease, a PCR (polymerase chain reaction) DNA analysis of peripheral blood lymphocytes (PBL) and of various tissues of healthy "at-risk", or ill baboons was performed. Proviral STLV/HTLV sequences were detected in all monkeys with symptoms of T-cell malignancy and/or antibodies to STLV-I/HTLV-I. For precise identification and characterization of the Sukhumi T-cell lymphoma virus, parts of the virus genome were mapped and sequenced from PCR derived fragments. A 420 nucleotide fragment of the env (gp 46) gene (analysed from 3 different DNA's) revealed 16.2% nucleotide divergence to the Japanese strain of HTLV-I and 14.8% to the Japanese strain of STLV-I including one deletion of a triplet. On the level of amino acid (a.a) sequence this revealed an exchange of 6 a.a. to STLV-I (4.3%), but only of 4 a.a. to HTLV-I (2.8%). The analysis of 120 nucleotides of the tax sequence (identical in 6 different DNAs) resulted in 5% nucleotide divergence to the HTLV-I (2.4% on the a.a. level) and 10% (7.3% a.a.) to the STLV-1. These results indicate that the Sukhumi T-cell lymphoma virus is a representative of the T-cell leukemia/lymphoma virus family, apparently more closely related to HTLV-I than to STLV-I genome. Furthermore, the infected monkeys from Sukhumi develop at a high rate a T-cell malignancy not observed among other baboons carrying STLV.

Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding.

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and E1 appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure.

A small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, is formed during processing of the polyprotein translated from the Sindbis virus subgenomic 26 S mRNA. In the accompanying paper we show that this 6K protein can be found in purified preparations of virions and that it is palmitoylated via thioester bonds with about four covalently bound fatty acids per molecule. To determine acylation sites on 6K and define a role for these fatty acids, we used site-directed mutagenesis to alter cysteine codons in the 6K gene of Sindbis virus cDNA. One of these mutants had a single cysteine replaced with a serine and the second had two adjacent cysteines replaced with an alanine-serine sequence. Transfection of the transcribed RNA from these two cDNAs produced infectious virus which contained 6K proteins that had decreased amounts of fatty acids. Intracellular formation and maturation of virus glycoproteins appeared to be unaffected by the mutations but the release of virus particles from mutant-infected cells was decreased about 70 to 90% from that observed with wild-type virus. Electron microscopy of virus-infected cells and of isolated virions showed that the 6K mutations led to large numbers of aberrant enveloped particles containing multiple nucleocapsids. These results indicate that the 6K protein and its state of acylation are important factors in Sindbis virus assembly and budding. Additional phenotypic changes are reported for virions released from cells infected with the mutationally altered viruses.

The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids.

A small hydrophobic polypeptide is encoded within the genome of the alphaviruses by a set of 165 nucleotides which map between the sequences for the two virus glycoproteins. This polypeptide has been referred to as 6K and was previously found on membranes in virus-infected cells. We report here that this protein is heavily acylated with long chain fatty acids covalently attached in hydroxylamine-sensitive ester bonds and that the 6K protein can be detected in purified preparations of virions. A polyclonal rabbit serum, raised against a peptide which contained the 16 amino acids at the amino-terminus of the 6K protein, was used to identify the 6K protein in infected cells and virions. This antibody also precipitated a 4K protein which was present in Sindbis virus-infected cells but not in virions. This latter protein was shown to be an underacylated form of the 6K protein and infected cells contained about twice as much 4K as 6K. In the cell there was close to a 1:1 stoichiometry between the 4K + 6K proteins and the virus glycoproteins E1, p62, and E2, but in virions the ratio of 6K to E1 + E2 ranged from 0.08 to 0.12.