Search

Temperature-sensitive mutants in the N complementation group of human adenovirus type 5 are defective at the nonpermissive temperature for replication of virus DNA and for transformation of rat embryo cells. We show that nuclear extracts prepared from Ad5ts 149-infected cells grown at the nonpermissive temperature fail to replicate DNA in vitro. The defect lies in the first step in the initiation of viral DNA synthesis, the formation of a covalent linkage between the terminal protein precursor (pTP) and dCMP. A 140 kilodalton (140 kd) protein which complements these defective extracts and contains DNA polymerase activity has been purified from HeLa cells infected with wild-type Ad2. It is tightly associated with the 80 kd pTP in a replication complex. Both of these proteins are products of the E2B region of the adenovirus genome, and the 140 kd protein coding sequences lie immediately downstream from those encoding the 80 kd protein. These results demonstrate that adenovirus encodes a novel DNA polymerase that is required for priming of DNA synthesis at the origin of replication. This protein may also function in the initiation of transformation of cultured cells.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.

An autoantibody known as anti-Jo-1 antibody is found in 25% of patients with myositis. Its prevalence in patients with both myositis and cryptogenic fibrosing alveolitis was 68% (13 out of 19 patients), compared with 7.5% in patients with myositis alone (four of 53) and 3% in patients with cryptogenic fibrosing alveolitis alone (two of 62). Anti-Jo-1 antibody may be useful in indicating patients with myositis and cryptogenic fibrosing alveolitis. Raynaud's phenomenon, the sicca syndrome, and mild arthritis are also often part of the syndrome.

Background Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. Results Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. Conclusion At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.