The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.

Considerable progress has been made over the past 10 years towards a full understanding of the functional significance of the structural changes resulting from the production of adenoviruses in permissive cells. Similarly, the host-virus interactions which are involved in viral replication and gene expression as well as in RNA nuclear export have been investigated. Post-embedding nonisotopic in situ hybridization has been proven to be a powerful tool for the study of nucleic acids in infected cells provided that controlled elimination of artifacts by appropriate treatments was undertaken. Adenovirus infected cells present two biological characteristics which could lead to false positive or negative results. First, they contain large amounts of single-stranded portions of viral DNA which are revealed with viral RNA molecules. Second, DNA-binding proteins are present which hide some nucleic acid sequences. By using a DNA probe and appropriate variations in the experimental protocol, it is possible to reveal specifically different kinds of targets, simultaneously single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), or only ssDNA, or only dsDNA, or only RNA. By using two probes labeled with different haptens, biotine and digoxigenin, it is possible to detect concomitantly two nucleic acid targets and, therefore, to study their relationships. In order to gain insight into the changes in the nucleus before cell lysis and to improve our knowledge on the series of steps leading to the release of adenoviruses from the nucleus, examination of cells at 41 h post-infection and identification of structures containing adenoviral nucleic acids were undertaken. In addition to the ultrastructural changes and precise distribution of cellular DNA and viral nucleic acid molecules already described in cells up to 24 h post-infection (for a review, see Puvion E, Puvion-Dutilleul F (1996) Exp Cell Res 229, 217-225), new results were obtained. Routine observation revealed the presence of: i) viruses in the cytoplasm, some being located next to nuclear pores; ii) abnormally large portions of the nuclear envelope devoid of underlying condensed chromatin; iii) proliferation of either the inner nuclear membrane only or both membranes of the nuclear envelope; and iv) electron-opaque grains in the nuclear compartment involved in viral genome transcription, and also in the clusters of interchromatin granules known to contain mature viral messenger RNA (Bridge E et al (1996) J Cell Biol 135, 303-314). In situ hybridization revealed the presence of: i) dsDNA in the cytoplasmic viruses indicating that they were mature viruses; ii) free viral dsDNA and ssDNA molecules in the cytoplasm whereas host DNA remained confined at the nuclear border; and iii) viral RNA in the newly-described electron-opaque grains we call, therefore, viral-RNA containing grains. Immunodetection of bromodeoxyuridine (BrdU) incorporated into DNA in pulse and pulse-chase experiments allowed us to ascertain that cells at 41 h post-infection were truly living cells and ...

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To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

Protein kinase activities copurifying with the 7200‐MrDNA‐binding protein of adenovirus on DNA‐cellulose chromatography and gel filtration in acrylamide/agarose have been partially characterized and purified. One of these kinases was found to phosphorylate efficiently the viral DNA‐binding protein in vitro and to be stimulated severalfold by the addition of histones, protamine, or polyamines. The kinase does not, however, phosphorylate histones, protamine, casein, or phosvitin. A second protein kinase was also recovered from single‐stranded DNA‐cellulose which is able to phosphorylate the 72000‐Mr DNA‐binding protein, but which is inhibited by the addition of histones. Phosphorylation in vitro of the 72000‐Mr DNA‐binding protein from the ts125 mutants of adenovirus by the histone‐stimulated protein kinase was found to be thermosensitive.

SUMMARYSeveral new polypeptides were detected in cells infected with CTHV, a cottontail rabbit herpesvirus. All of them (Mr 150K, ll0K, 93K, 83K, 75K and 35K) accumulated in the nucleus during the infectious cycle, and all except the 150K species bound to DNA-cellulose affinity columns in low-salt buffers. Polyclonal antisera prepared against the 35K DNA-binding protein also recognized the 75K species. Although the 75K protein could be detected earlier in infection than the 35K protein, late in the infectious cycle the latter increased to an abundance approaching that of cellular histones. Treatment of partially purified virions with a non-ionic detergent indicated that the 35K protein, but not the 75K protein, is a component of capsid/ tegument structures. The anti-35K/75K serum did not cross-react with herpesvirus sylvilagus virion proteins, which, in an electrophoretic comparison, exhibited both similarities to and differences from the virion proteins of CTHV. Labelling of CTHVinfected cells with [32p]orthophosphate revealed the presence of phosphoproteins electrophoretically comigrating with the 93K, 83K, 75K and 35K proteins.

A DNase activity was found associated with isolated components of adenovirus types 5 and 7. The enzymatic activity was associated with the purified virus and viral components extracted from the soluble material of infected cells. The DNase activity of adenovirus type 5 was maximum at pH 5.5 and 7.0 whereas adenovirus 7 was active only at pH 7.0. Both DNases were shown to be located in the penton fraction.

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