Development of an in vitro mRNA degradation assay utilizing extracts from HIV-1- and SIV-infected cells.

We previously demonstrated that cellular mRNAs are degraded in CD4 positive lymphocytes infected by the human immunodeficiency virus, HIV-1, but not in cells infected by the simian lentivirus, SIV. To begin to define the molecular mechanisms underlying this RNA degradation, we have established an in vitro RNA degradation assay utilizing extracts from both infected and uninfected cells. We found that in vitro transcribed, 32P-radiolabeled actin RNA was degraded in extracts prepared from CEM, CEMx174, and C8166 cells which were infected with HIV-1. Minimal actin RNA degradation was observed in extracts prepared from uninfected cells. Similarly little degradation was observed in cell-free extracts prepared from SIV-infected cells. To determine if viral RNA sequences could impart enhanced stability to cellular RNAs in our in vitro assay, we prepared radiolabeled RNAs that contained selected viral RNA determinants. One such RNA contained the HIV-1 specific TAR (transactivating region) sequence (nucleotides 1-111) appended to a reporter CAT RNA. Like the cellular actin RNA, these TARCAT RNAs were degraded in HIV-1-infected cell extracts, but not in extracts from uninfected cells or extracts prepared from SIV-infected cells. In contrast, an RNA containing only authentic HIV-1 sequences comprising TAR and gag sequences was more stable than actin RNA in HIV-1-infected extracts. These results, taken together, suggest that the in vitro assay reproduces events that occur in vivo and provide a starting point for identifying the factor responsible for cellular RNA degradation in HIV-1-infected cells.

Interferon treatment inhibits virus replication in HIV-1- and SIV-infected CD4+ T-cell lines by distinct mechanisms: evidence for decreased stability and aberrant processing of HIV-1 proteins.

We have examined the effects of interferon (IFN)-alpha/beta on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse-chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.

Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain.

Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.

In vitro responses to Leishmania antigens by lymphocytes from patients with leishmaniasis or Chagas' disease.

T cell responses are correlated with recovery from and resistance to leishmaniasis. Antigens of Leishmania chagasi were evaluated by determining their ability to elicit in vitro proliferation and cytokine production in peripheral blood lymphocytes and in T cell lines and clones from patients with histories of leishmaniasis or Chagas' disease. Antigens tested were selected by their reactivity with patient antibodies. Several of the antigens induced proliferative responses in peripheral blood lymphocytes from patients recovered from visceral or cutaneous leishmaniasis or with chronic Chagas' disease. Two purified glycoproteins, 30 and 42 kD, were consistently among the most effective in eliciting high proliferative responses and IL-2 production. Lymphocytes from a recovered visceral leishmaniasis patient were used to produce T cell lines against either the 30- or 42-kD antigen. Each of the lines responded to both of these antigens as well as to crude leishmania lysate. CD4+ T cell clones specific for either or both of these antigens were also isolated from a visceral leishmaniasis patient. In contrast, rabbit antisera produced against these two antigens were not crossreactive. Both antigens were effective in inducing the production of IFN-gamma from T cell lines from both leishmaniasis and Chagas' disease patients. These studies demonstrate the potential for defining parasite antigens with broad immunostimulatory capabilities.