The accessory Vpr protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators.

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

We report that p160 nuclear receptor coactivators potentiate the transactivating activity of Tat, the most potent virally encoded transactivator of HIV-1. One of the p160 proteins (GRIP1) is tethered to the HIV-1 long terminal repeat (LTR) through B-responsive elements, most likely via NF-B, with which it also associates through its coactivator motifs (LXXLL motifs, "NR boxes"). Indeed, the Tat-stimulated B-defective HIV-1 LTR had a markedly impaired response to GRIP1, whereas NR box-defective GRIP1 proteins lost part of their Tat coactivator effect on the HIV-1 LTR. Through its N-terminal basic helix-loop-helix and C-terminal domains, GRIP1 binds to the N-terminal region of Tat and to the host cell protein cyclin T1, respectively, which is normally complexed with CDK9 as P-TEFb. Thus, NF-B is crucial for tethering p160 coactivator molecules to the HIV-1 LTR, allowing full activation of this promoter by Tat. Interestingly, cotransfection of Tat, GRIP1, and cyclin T1 enhanced not only the activity of the HIV-1 LTR, but also the glucocorticoid receptor-mediated stimulation of the mouse mammary tumor virus (MMTV) promoter, suggesting that Tat can also attract the P-TEFb complex to the MMTV LTR through GRIP1. Thus, it appears that the coactivator complexes of the HIV-1 and MMTV LTRs both include p160 coactivators and use similar coactivator and elongation complexes for their transcription. Tat may function as an adaptor molecule, efficiently stimulating the processes of transcription initiation and elongation through potentiation of the coupling of p160 coactivators and the P-TEFb complex.

Understanding the orchestrated genome-wide cellular responses is critical for comprehending the early events of coronavirus infection. Microarray analysis was applied to assess changes in cellular expression profiles during different stages of two independent, highly controlled murine hepatitis virus (MHV) infections in vitro. Fibroblast-like L cells were infected at high multiplicity in order to study the direct effects of a synchronized lytic coronavirus infection. Total RNA was harvested from MHV-or mock-infected L cells at 3, 5 and 6 h post-infection and hybridized to Affymetrix microarrays representing approximately 12 500 murine genes and expressed sequences. The expression data were compared to their respective mock-infected controls. Quantitative RT-PCR of selected transcripts was used to validate the differential expression of transcripts and inter-experiment reproducibility of microarray analysis. It was concluded that MHV-A59 infection in fibroblast-like cells triggers very few transcriptional cellular responses in the first 3 h of infection. Later, after having established a productive infection, a chemokine response is induced together with other cellular changes associated with RNA and protein metabolism, cell cycle and apoptosis. Interferon responses are not triggered during infection, although the L cells can be readily stimulated to produce interferon by dsRNA, a known potent inducer of interferon. Possibly, the interferon response is actively counteracted by a virus-encoded antagonist as has been described previously for other RNA viruses.

Poliovirus mutants with extended (> 150-nt) deletions in the 5'-untranslated region between the internal ribosome entry site and the initiator codon have been selected previously (Pilipenko et al., Cell 68, 119-131, 1992; Gmyl et al., J. Virol. 67, 6309-6316, 1993). These deletions were transferred into the genome of a mouse-pathogenic poliovirus strain and found to be strongly attenuating. The deletions can be considered as covering three structural elements, a stem-loop (domain E) with a conserved cryptic AUG and two spacers, upstream and downstream of it. In an attempt to identify putative essential determinants of neurovirulence in these individual structural elements, appropriate mutants were engineered. The results demonstrated that neither of the above elements is essential for neurovirulence. The results strongly suggested that the presence of a cryptic AUG in the oligopyrimidine/AUG tandem followed, at a sufficient distance, by the initiator codon was necessary to ensure the neurovirulent phenotype of our constructs. On the other hand, the attenuated phenotype appeared to correlate with the occurrence of the initiator AUG as a moiety of the oligopyrimidine/AUG tandem. Possible mechanisms underlying these effects are discussed. Identification of the cryptic AUG as an essential determinant for neurovirulence provides a rational basis for the design of genetically stable attenuated poliovirus variants.

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Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10 7 to 10 8 PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.Coronaviruses belong to the Coronaviridae family in the Nidovirales order. Coronavirus infections can follow different scenarios in vivo, as well as in vitro, suggesting complex and diverse interaction with their hosts. Viral genome expression and molecular mechanisms of virus-host interaction have mostly been studied in the context of efficient coronavirus replication in a lytic infection of cultured, transformed cells. Cytopathic infection with one of the best-studied coronaviruses, murine hepatitis virus (MHV), causes drastic changes in the cellular infrastructure (20, 36) and metabolism (14,37,44) and results in cell death. Coronavirus infection in vitro can take a different course and exhibit limited or no cytopathic effect (CPE) or virus-induced cell death. Productive noncytopathic infections have been described for primary cells or relatively differentiated cell lines infected with coronaviruses MHV (19,21,27), human coronavirus OC43 (1), human coronavirus 229E (2), and severe acute respiratory syndrome (SARS) coronavirus (5). Sturman and Takemoto reported that MHV infection of the mouse BALB/3T3 cell line produced high yields of virus but showed little CPE within 1 day postinfection (p.i.) (40). Mouse embryo fibroblast BALB/3T3 cell lines are obtained in such a way that the cells exhibit the contact inhibition that is characteristic of nontransformed cells in vivo (41). The coronavirus CPE became pronounced when BALB/3T3 cells were transformed, either spontaneously or by infection with oncogenic viruses (40). Which mechanisms or host factors prevent CPE and virus-induced cell death in nontransformed ...

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is the sole known receptor of murine hepatitis virus (MHV) A59, but the available, often qualitative, data about CEACAM1 expression does not explain MHV organ tropism. Ceacam1 transcripts undergo alternative splicing resulting in multiple isoforms, including secreted CEACAM1 isoforms that can neutralize the virus. We determined the quantities of Ceacam1 transcripts encoding membranebound and secreted isoforms in mouse organs and a set of cell lines. In vivo, the lowest receptor mRNA levels were found in brain and muscle and these were similar to those in easily infectable cultured cells. While the quantities of the receptor transcripts varied between mouse organs, their abundance did not correlate with susceptibility to MHV infection. The proportion of transcripts encoding secreted isoforms also could not explain the selection of sites for virus replication, as it was constant in all organs. Our data suggest that neither of the two CEACAM1 isoforms defines MHV organ tropism.