Lack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins.

All lentiviruses contain an open reading frame located shortly upstream or inside of the env gene and encoding a small protein which has been designated Tat. This designation was mainly with respect to the positional analogy with the first exon of the trans-activator protein of the well studied human immunodeficiency virus type 1 (HIV-1). In this work we comparatively studied the trans- activation activity induced by Tat proteins of the small ruminant Maedi Visna virus (MVV) of sheep and Caprine arthritis encephalitis virus (CAEV) of goats on MVV and CAEV LTRs with that induced by the human lentivirus HIV-1 on its own LTR. The HIV-1 LTR alone weakly expresses the reporter GFP gene except when the HIV-1 Tat protein is coexpressed, the GFP expression is increased 60-fold. In similar conditions only minimal trans-activation increasing two- to three-fold the MVV and CAEV LTR activity was found with MVV Tat protein, and no trans-activation activity was detected in any used cell type or with any virus strain when CAEV Tat was tested. These results indicate that the small ruminant lentiviruses (SRLV) differ from the primate lentiviruses in their control of expression from the viral LTRs and put into question the biological role of the encoded protein named "Tat."

Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages.

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.

Visna/maedi virus Env protein expressed by a vaccinia virus recombinant induces cell-to-cell fusion in cells of different origins in the apparent absence of Env cleavage: role of glycosylation and of proteoglycans.

The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV- env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV- env-MV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV- env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, beta-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA.

Properties of wild-type, C-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral vector particles.

We have characterized the properties of the maedi-visna virus (MVV) glycoprotein, which has a long cytoplasmic C-terminal domain, and of a panel of C-terminally truncated and C-terminally chimeric MVV-Env constructs. Cells expressing wild-type MVV glycoprotein form syncytia with target cells from many different species and tissues, demonstrating that the MVV-Env cellular receptor is widely distributed. Similar to the situation with other lentiviral glycoproteins, truncation of the C-terminal domain of MVV-Env significantly increases its membrane fusion capacity. However, despite their presence in a fusogenic form at the cell surface, neither the wild-type nor any of the C-terminally modified MVV-Env constructs, these latter lacking sterically inhibitory C termini, were able to successfully pseudotype murine leukemia virus- or human immunodeficiency virus-derived vector particles.

Temperature inducible beta-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family.

The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of beta-structure with rising temperatures in all activation domain peptides. The high stability of beta-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce alpha-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L78E79-->D78L79) showed wild-type structure, the M32 mutant peptide (L78L81L83-->A78A81A83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a beta-structural element is involved in binding to a cellular cofactor.

Analysis and PCR detection of antigen compositions of ovine progressive pneumonia virus.

Ovine progressive pneumonia virus (OPPV) was proliferated utilizing sheep foetal lung cells, and the cytopathic effect (CPE) of the virus was investigated. OPPV was purified with a 10%-sucrose cushion and then with 20%-55% discontinuous sucrose density gradient centrifugation. The structural proteins and antigen compositions of OPPV were analysed by SDS-PAGE and Western blotting. Besides, the OPP proviral cDNAs of the virus-infected cell cultures and the peripheral blood monocytes from sheep infected by the virus were detected using polymerase chain reaction (PCR). The results show that the CPE of sheep foetal lung cells infected by OPPV is typical of the disease. The purified virions are intact and of high purity when observed with an electron microscope. OPPV proteins consist of 18 polypeptide bands and the molecular weights range from 18 to 120 kd. Among these, 3 were glycoproteins (designated by gp120, gp50 and gp47). The appearance and peak time of the p28 antibody from sheep inoculated with OPPV are earlier than those of the p94 antibody to OPPV. Besides, the strength of immune reaction of p28 antibody is greater than that of p94 antibody. With PCR, it is demonstrated that the initial time for OPP proviral cDNAs to be integrated into the sheep foetal lung cells and the peripheral blood monocytes in sheep were 24 h and 9 d after inoculation, respectively.

The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines.

The HIV and visna lentiviruses induce an inflammatory reaction in the central nervous system (CNS) of the infected hosts leading to dysmyelination, demyelination, and neuronal loss. The basic domain of the transactivating Tat protein has been involved in CNS damage. Infusion of basic containing domain Tat peptides in the lateral ventricle (systemic injection) or in the grey matter, i.e., hippocampus and thalamus (local injection), induced an inflammatory process characterized by the formation of an edema and invasion of macrophage accompanied by reactive astrogliosis. Control peptides originating from either lentiviral proteins or irrelevant protein as ovalbumin did not lead to any inflammatory reaction or cell death. The inflammation led to the loss of ependymal cells in the lateral ventricles and neurons in the grey matter. RNA extracted from the Tat-injected hemisphere reacted with TNF-alpha, IL-1 alpha and beta, and IL-6 probes. The macrophage/microglia inducible nitric oxyde synthase was also expressed. Blockade of TNF-alpha by a pentoxifylline treatment led to the decrease of IL-1 and iNOS expression accompanied by a reduction of the volume of the lesions indicating that the Tat-induced lesions might be mediated by TNF production.

The Rev protein of visna virus is localized to the nucleus of infected cells.

Visna virus is a lentivirus of sheep that is distantly related to the human lentivirus HIV-1. Like other lentiviruses, the genome of visna virus contains multiple small open reading frames that encode viral regulatory proteins. The product of one of these regulatory genes is the visna virus Rev protein, Rev-V. In this report, immunoprecipitation of visna virus-infected cells using a specific anti-Rev-V antibody, generated to a synthetic, carboxyl-terminal peptide of Rev-V, brings down a 22.5-kDa protein identical in size to the protein expressed from a functional Rev-V cDNA clone. Examination of the phosphorylation state of Rev-V indicates that it, unlike the Rev proteins of HIV-1 and CAEV, is not efficiently phosphorylated in infected cells. Cell fractionation and immunofluorescence analysis indicate that, in contrast to a previous report, Rev-V is strongly localized to the nucleus and concentrated in nucleoli of visna virus-infected cells. In addition, Rev-V localizes similarly in several different primary cells, in particular macrophages, infected with visna virus. These data indicate that the Rev-V protein is produced during visna virus infection and is localized to the nucleolus of the infected cell.