Previous studies have shown that infection of human fibroblasts with human cytomegalovirus (HCMV) results in activation of cellular interferonresponsive gene expression. We demonstrate here that infection of human fibroblasts with herpes simplex virus type 1 (HSV-1) in the absence of de novo protein synthesis also induces the expression of interferon-responsive genes. Five genes tested (encoding ISG54, IFI56, ISG15, 9-27 and MxA) were activated by infection with HSV-1, although the degree of response varied between the individual genes. HSV-1 was a less efficient inducer than HCMV. The effect was a consequence of binding of the virus particle to the cell surface or of the presence of virion components within the infected cell. Induction was mediated by a pathway other than the mechanism through which interferon-α mediates its effects on cellular gene expression.

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The recombinant virus assay (RVA) is a method for assessing the susceptibility of human immunodeficiency virus type 1 (HIV-1) plasma isolates to antiretroviral drugs. The RVA involves the production of viable virus in vitro by homologous recombination of RT-PCR products from plasma virus with a noninfectious reverse transcriptase (RT) or protease (PR)-deleted cloned HIV-1 provirus. In this study, we have constructed RVA plasmids with contiguous deletions in RT, PR, and the p7/p1 and p1/6 gag protease cleavage sites (CS). The deletions in these plasmids allow generation of recombinant viruses with all loci currently identified as important for resistance to anti-HIV-1 drugs being derived from the clinical isolate, including CS mutations that compensate for the reduced fitness of viruses resistant to protease inhibitors (Doyon et al., J Virol 1996:70:3763-3769). We have also used these new constructs to generate viruses with or without compensatory CS mutations, and examined the effects on fitness. In the case of an indinavir-selected virus, fitness was restored close to that of a wild type virus when a vector deleted in the CS and PR was used. With an amprenavir-selected isolate, virus fitness was incompletely restored by including the CS, and this defect appeared to be partially due to reduced infectivity of the virions. We conclude that the CS mutations were required for optimum detection of resistance in the RVA, but that virus fitness can remain compromised even in the presence of compensatory CS mutations.

Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift at the junction of the gag and pol coding sequences.Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6 gag L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency. INTRODUCTIONExpression of the HIV-1 pol gene requires a programmed 21 ribosomal frameshift event (Jacks et al., 1988). Most ribosomes translating the viral genomic RNA terminate at the gag stop codon, generating the Gag polyprotein, encoding structural proteins. However, a shift of about 5-10 % into the overlapping pol frame in response to the frameshift signal yields the Gag-Pol fusion protein from which pol-encoded proteins viral protease (PR), reverse transcriptase (RT) and integrase are liberated by PRmediated proteolysis. The frameshift signal of HIV-1 has two components, a slippery sequence, UUUUUUA, where the frameshift takes place, and a stimulatory RNA secondary structure immediately downstream. The precise structure of the HIV-1 stimulatory RNA has been the subject of debate (Jacks et al., 1988;Le et al., 1991;Du et al., 1996; Dinman et al., 2002;Dulude et al., 2002;Staple & Butcher, 2003; reviewed by Brierley & Dos Ramos, 2005), but from mutational analysis, secondary structure probing and nuclear magnetic resonance (NMR) analysis, the active element is most probably a stem-loop consisting of a twostem helix capped by a tetraloop (Dulude et al., 2002;Staple & Butcher, 2005;Gaudin et al., 2005; see Fig. 1). Frameshifting is crucial to HIV replication, as expression of Gag-Pol allows targeting of replicative enzymes to the particle core during assembly. Further, it sets a precise ratio of Gag : Gag-Pol, the maintenance of which appears to be essential (Park & M...

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