Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

Programmed ؊1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses, including human immunodeficiency virus type 1 (HIV-1), to produce multiple proteins from a single mRNA. ؊1 PRF induces a subset of elongating ribosomes to shift their translational reading frame by 1 base in the 5 direction. The appropriate ratio of Gag to Gag-Pol synthesis is tightly regulated by the PRF signal which promotes ribosomes to shift frame, and even small changes in PRF efficiency, either up or down, have significant inhibitory effects upon virus production, making PRF essential for HIV-1 replication. Although little has been reported about the cellular factors that modulate HIV-1 PRF, the cis-acting elements regulating PRF have been extensively investigated, and the PRF signal of HIV-1 was shown to include a slippery site and frameshift stimulatory signal. Recently, a genome-wide screen performed to identify cellular factors that affect HIV-1 replication demonstrated that down-regulation of eukaryotic release factor 1 (eRF1) inhibited HIV-1 replication. Because of the eRF1 role in translation, we hypothesized that eRF1 is important for HIV-1 PRF. Using a dual luciferase reporter system harboring a HIV-1 PRF signal, results showed that depletion or inhibition of eRF1 enhanced PRF in yeast, rabbit reticulocyte lysates, and mammalian cells. Consistent with the eRF1 role in modulating HIV PRF, depleting eRF1 increased the Gag-Pol to Gag ratio in cells infected with replication-competent virus. The increase in PRF was independent of a proximal termination codon and did not result from increased ribosomal pausing at the slippery site. This is the first time that a cellular factor has been identified which can promote HIV-1 PRF and highlights HIV-1 PRF as essential for replication and an important but under exploited antiviral drug target.The ability of ribosomes to maintain the correct translational open reading frame (ORF) 2 is fundamental to the integrity and fidelity of protein synthesis. However, there are a number of examples in which elongating ribosomes are programmed to shift their translational ORF 1 base in either the 5Ј or 3Ј direction (Ϫ1 or ϩ1 ribosomal programmed ribosomal frameshift (PRF), respectively) to translate multiple proteins from a single mRNA (1-5). HIV-1 is one example of a virus that uses this process as an integral part of its replication cycle.The HIV-1 Gag and Pol proteins are synthesized as p55 Gag or p160Gag-Pol polyprotein precursors using the same translation start codon but different ORFs in the full-length viral mRNA (6, 7). The gag ORF is located at the 5Ј end of the viral mRNA, whereas the pol ORF is 3Ј of the HIV-1 PRF element and outof-frame with the gag ORF (Fig. 1). Therefore, the enzymatic protein products of the pol gene are only translated through a Ϫ1 PRF event (8 -10). Importantly, the frequency of the PRF is controlled precisely via the interaction between viral RNA cisacting elements and host translation factors. The frequency of the PRF in HIV-1...

A significant difference in the recombination rates between human immunodeficiency virus type 1 (HIV-1) and the gammaretroviruses was previously reported, with the former being 10 to 100 times more recombinogenic. It is possible that preferential copackaging of homodimers in the case of gammaretroviruses, like murine leukemia virus (MLV), led to the underestimation of their rates of recombination. To reexamine the recombination rates for MLV, experiments were performed to control for nonrandom copackaging of viral RNA, and it was found that MLV and HIV-1 exhibit similar crossover rates. The implications for control of proviral ploidy and preferential recombination during minus-strand DNA synthesis are discussed.It is not surprising that retroviruses can efficiently undergo homologous recombination since the virions are diploid, providing the opportunity to recombine during reverse transcription. Moreover, two obligatory strand transfers occur during reverse transcription, so the type of template-switching ability that would be required for recombination is built into the viral life cycle. Results from our laboratory and from others also indicate that retroviral recombination occurs predominantly during minus-strand synthesis using viral RNA as a template (2,10,(28)(29)(30).Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) recombines at a rate of at least three crossovers per cycle of replication in HeLaT4 cells (30). It was reported to be higher in other cell types, including primary cells and established cell lines. For macrophages, it was reported to be an astounding 30 crossovers per genome per cycle of replication (13), although this has been recently called into question (14). It was found through in vivo studies that the mutation rates of HIV-1, spleen necrosis virus, and murine leukemia virus (MLV) were quite similar (21,24,25), and the strand transfer activities of HIV-1 and MLV were found to be equivalent on the basis of measuring the rates of homologous recombination between direct repeats within the same genome (17). However, the intermolecular recombination frequencies between separate, copackaged viral RNAs were reported to be significantly higher for HIV-1 than for MLV (10). For MLV, the recombination rate was calculated to be 0.3 to 0.4 crossovers per cycle of replication, which is about 10-fold lower than what we and others found for HIV-1 (2, 10, 30). One of the assumptions previously made for measuring recombination rates was that the two genetically distinct viral RNAs were randomly copackaged into virions. However, this was recently reported not to be the case for MLV (7, 11). Instead, there is preferential copackaging of homodimers, which likely led to an underestimation of the MLV recombination rate since the recombinant proviral progeny of homodimeric virions would not be scored (17). In this report, we sought to determine the MLV rate of crossover by exclusively examining the progeny of heterodimeric virions.Strategy for analyzing proviral progeny of heterozygou...

Retinoids are labile compounds with less than micromolar solubility in aqueous solutions. They are normally associated with a carrier protein, interphotoreceptor retinoid binding protein, in the interstitial spaces of the retina. In the past, experimental retinoids have been delivered to isolated retinas and photoreceptors using either unprotected ethanolic suspensions or phospholipid vesicles that protect but require burdensome preparation. The objective of these experiments was to characterize the protection of retinoids and their delivery to isolated photoreceptor cells at high concentrations using fatty-acid-free bovine serum albumin (BSA) as a convenient vehicle and protectant. Our results confirm that BSA is a useful protectant and vehicle for delivery of retinoids to isolated retinas and photoreceptors.

Retinoids are labile compounds with less than micromolar solubility in aqueous solutions. They are normally associated with a carrier protein, interphotoreceptor retinoid binding protein, in the interstitial spaces of the retina. In the past, experimental retinoids have been delivered to isolated retinas and photoreceptors using either unprotected ethanolic suspensions or phospholipid vesicles that protect but require burdensome preparation. The objective of these experiments was to characterize the protection of retinoids and their delivery to isolated photoreceptor cells at high concentrations using fatty-acid-free bovine serum albumin (BSA) as a convenient vehicle and protectant. Our results confirm that BSA is a useful protectant and vehicle for delivery of retinoids to isolated retinas and photoreceptors.