Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed ؊1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.Due to their compact genomes, viruses often develop novel modes of gene expression in order to synthesize multiple proteins from a single RNA species. For example, human immunodeficiency virus type 1 (HIV-1) uses a unique mode of gene regulation termed programmed Ϫ1 ribosomal frameshifting. The structural and enzymatic components of the virus are synthesized as polyproteins with a common N terminus. The open reading frame (ORF) encoding the structural 55-kDa Gag protein is located at the 5Ј end of the mRNA. The 160-kDa Gag-Pol polyprotein is translated only as a result of a programmed Ϫ1 ribosomal frameshift event (Fig. 1A) (17). The pol ORF, which encodes the enzymatic proteins, including viral protease (PR), reverse transcriptase, and integrase, is 3Ј to the gag gene and out of reading frame with respect to the gag ORF. Programmed Ϫ1 ribosomal frameshifting allows synthesis of the required ratio of Gag to Gag-Pol, which is highly regulated in retroviruses, with frameshifting occurring at levels of 5 to 10% for HIV-1 (17, 30). These polyprotein precursors are incorporated into viral particles and are subsequently cleaved during viral maturation by the viral PR.In vitro and in vivo studies demonstrate that there are two cis-acting elements located within the overlapping region of the HIV-1 gag and pol genes that are critical for translational frameshifting to occur (Fig. 1B). The first is the heptamer UUUUUUA, where frameshifting occurs and which computer analysis indicates is invariant (see Results). The second is a structural RNA motif downstream of the heptamer that assumes a stem-loop and possibly a pseudoknot structure (9,11,12,26). An 8-nucleotide spacer separates these two regions.Other viruses as well as additional retroviruses also employ ribosomal frameshifting during viral gene expression (3, 12). The consensus viral slippery sequence is X XXY YYZ, where spaces indicate the ...

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified (ϳ10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach. (HEPATOLOGY 2003;37:994-1005.)

Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cellbased system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4 ؉ T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.The ability of human immunodeficiency virus type 1 (HIV-1) to establish a latent infection results in life-long virus persistence even after long-term antiretroviral therapy (ART). 4 The role that latency plays in preventing sustained clearance of the virus infection has become evident in recent years. Patients that have been successfully treated with ART, having undetectable levels of viral RNA (below 50 copies/ml) in the plasma for years, experienced rapid virus rebound upon withdrawal of therapy (1, 2). Moreover, it was found that after T cell activation, virus could be isolated from CD4 ϩ T cells taken from these patients, underscoring the need to eliminate the latently infected cells to eradicate the virus (3-5).Activation of latent proviruses from infected cells in combination with ART is part of a therapeutic strategy that may lead to the complete elimination of HIV infection. Prior attempts to "flush out" the virus by activation of latently infected resting CD4 ϩ T cells with the administration of IL-2 and/or anti-CD3 monoclonal antibodies were ultimately unsuccessful, probably because of its inability to reach all of the latent viral reservoirs and the toxicity of the regimen (6 -10). A more promising approach to complete viral clearance is the use of small molecules with pharmacological properties that allow them to access the viral reservoirs and to specifically reactivate the latent proviruses. The concept of small molecule activation of latent HIV-1 has been tested in a clinical study using the histone deacetylase (HDAC) inhibitor valproic acid (VA) (11). However, it is questionable whether VA alone can be used as a supplement to ART for succe...