The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-B. NF-B is retained in the cytoplasm through binding to its inhibitor IB␣. The critical step in NF-B activation is the phosphorylation of IB␣ by the IB kinase (IKK) complex. This activity releases NF-B from IB␣ and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-B in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2␣-binding domain, can also activate NF-B. This result indicated that NF-B activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2␣ relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR 0/0 mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKK␤ subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-B essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-B signaling pathway without using its kinase activity.

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

Ribosomal frameshifting on retroviral RNAs has been proposed to be mediated by slippage of two adjacent tRNAs into the -1 direction at a specific heptanucleotide sequence. Here we report a computer-aided analysis of the structure around the established or putative frameshift sites in a number of retroviral, coronaviral, toroviral, and luteoviral RNAs and two dsRNA yeast viruses. In almost all cases a stable hairpin was predicted four to nine nucleotides downstream of the shifty heptanucleotide. More than half of the resulting hairpin loops give rise to potential pseudoknotting with sequences downstream of this hairpin. Especially in the case of the shifty heptanucleotides U UUA AAC and G GGA AAC, stable downstream pseudoknots are present. Indications were also found for the presence of pseudoknots downstream of amber stop condons at readthrough sites in some retroviral RNAs.

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Human immunodeficiency virus type 1 plasma viruses from 29 entry inhibitor-naive patients were characterized for their susceptibilities to T-20, AMD3100, and RANTES. A strikingly wide range of susceptibilities to T-20 was observed that was influenced by coreceptor usage but not by the susceptibilities of the viruses to inhibitors that target the chemokine receptors or by polymorphisms in the gp41 N helix.The human immunodeficiency virus type 1 (HIV-1) entry process (2,19,27) can be inhibited by several drugs (3,14,22,23), which belong to three groups according to the step they inhibit: (i) inhibitors of the interaction between the viral surface glycoprotein (gp120) and CD4, which target the CD4-binding site on gp120; (ii) inhibitors of the interaction between gp120 and CCR5 or CXCR4 (e.g., chemokines and their derivatives or small organic molecules that antagonize chemokine receptor activity); and (iii) fusion inhibitors, which are peptides derived from the sequence of the viral transmembrane glycoprotein (gp41) that prevent the formation of a hairpin structure required for membrane fusion. One of these peptides, T-20 (enfuvirtide), is currently being evaluated in phase III clinical trials (15,25,26). The optimization of treatment strategies that include entry inhibitors will rely on the availability of methods capable of determining baseline viral susceptibility and acquired resistance to these drugs. In addition, the characterization of the determinants of baseline susceptibility and of acquired resistance to entry inhibitors may provide valuable information on the viral entry process and on the precise mechanism of action of these drugs.We have developed a recombinant virus assay that permits the assessment of viral susceptibility to entry inhibitors. We modified a pNL4-3 molecular clone by deleting the region of the envelope gene encoding gp120 and the ectodomain of gp41 (positions 6480 to 8263) and replacing it with a linker that contains a unique MluI restriction site (vector 43-⌬env). Recombinant virus was produced by cotransfection of 293-T cells with the MluI-linearized 43-⌬env vector and a reverse transcription-PCR product, amplified from patient plasma samples, which encompasses the deleted region and carries short overlaps that allow homologous recombination. Virus-containing supernatants were used to infect subconfluent U373MG-CD4 cells expressing either CCR5 or CXCR4 (17), in the absence or in the presence of increasing concentrations of entry inhibitors. These target cells carry an HIV-1 long terminal repeat-lacZ cassette, which allows the quantification of single cycle infectivity by a colorimetric assay based on HIV-1 Tat-induced expression of ␤-galactosidase (24). The concentrations inhibiting 50% of virus infectivity (IC 50 s) were calculated by using the median-effect equation (6).The recombinant virus assay was first used to determine the baseline susceptibilities of subtype B primary viruses to the fusion inhibitor T-20 (American Peptide Company, Inc., Sunnyvale, Calif.). Plasma samples sel...