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Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors.

Vascular endothelial cells are a target for blood-borne pathogens which may affect their integrity and thromboresistant properties. Here, we report that cultured bovine and human endothelial cells lose their thromboresistance following interaction with the avian hemangioma-inducing retrovirus. We show that the envelope (env) gene product, glycoprotein 85, is responsible for this effect, which appears soon after infection without viral replication or cell transformation. Induction of thrombogenicity is associated with a reduction in prostacyclin release and increased expression of tissue factor. These observations may explain the occurrence of thrombosis frequently observed in association with the hemangiosarcomas induced by avian hemangioma-inducing retrovirus. These unique endothelial cell-virus interactions may also be a model for the pathogenesis of various vascular diseases.

High doses of aqueous protein antigens induce a form of immunological tolerance in which interleukin 2 (IL-2)- and interferon gamma (IFN-gamma)-secreting T helper type 1 (Th1) cells are inhibited, but IL-4-secreting (Th2) cells are not. This is manifested by reduced proliferation of antigen-specific T cells upon in vitro restimulation, and marked suppression of specific antibody responses of the immunoglobulin (Ig)G2a, IgG2b, and IgG3 isotypes, but not of IgG1 and IgE. The role of the immunomodulatory cytokine IL-4 in this model of unresponsiveness to protein antigens has been examined. Administration of tolerizing antigen itself primes splenic CD4+ T cells for secretion of lymphokines, both IL-2 and IL-4. Neutralization of IL-4 in vivo with the anti-IL-4 antibody 11B11 during tolerance induction augments IFN-gamma production by T cells of tolerant mice, and reverses the suppression of IgG2a, IgG2b, and IgG3. This blockade of IL-4 function does not, however, restore the proliferative responses of T cells, suggesting that reduced T cell proliferation is due to direct T cell inactivation or anergy. Inhibiting the activity of IL-4 in vivo also inhibits the expansion of antigen-specific Th2-like cells, which are resistant to the induction of unresponsiveness. Thus, the immunologic consequences of high-dose tolerance are due to a combination of clonal T cell anergy and IL-4-mediated immune regulation.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.

We isolated a field strain of avian hemangioma retrovirus (AHV) which induces a cytopathic effect (CPE) on cultured avian and mammalian cells shortly after infection. The kinetics of cell killing were dependent on the multiplicity of infection. The CPE on avian and mammalian cells was independent of virus replication, because UV-irradiated virus led to cell death as well. Biochemical and genetic experiments indicated that AHV env gene products were responsible for the CPE. Partially purified AHV envelope glycoproteins (gp85), but not those of the Rous sarcoma virus Prague C strain, induced a CPE. Rous-associated virus type 1, in which the env region was replaced by the AHV gp85 region, induced a CPE on avian and mammalian cultured cells. Therefore, we suggest that CPE is induced by AHV via interaction between viral gp85 and the cell membrane. This mode of CPE is unique among avian sarcoma-leukemia viruses.