APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.Replication of human immunodeficiency virus type 1 (HIV-1) in primary cells is dependent on the expression of Vif protein, which counteracts the activity of the host cytidine deaminases APOBEC3G (APO3G) and APOBEC3F (4,25,29,32). In the absence of Vif, APO3G is incorporated into virus particles (11,16,19,20,26,27,30), resulting in hypermutation of the viral genome (19) or degradation of mutated cDNA (14, 18, 31) via a DNA repair mechanism (reviewed in references 3 and 12). Interestingly, human APO3G is not only packaged into human immunodeficiency viruses but also incorporated into simian immunodeficiency viruses and murine leukemia virus (9,18,19). Packaging of APO3G into such diverse viruses suggests that it either is a relatively nonspecific process or involves signals shared by these viruses. APO3G can bind RNA in vitro (10). Indeed, several reports have noted that the presence of viral RNA enhanced APO3G encapsidation (28); however, others (17, 23) suggested that viral RNA was not essential for APO3G packaging (2,5,8,17,23,28).To further study the role of viral RNA in the packaging of APO3G into HIV-1 virions, we first compared the packaging of APO3G into either the wild-type infectious NL4-3 virus or a helper virus (C-Help) whose RNA genome is not packaged due to a 33-base deletion in the putative RNA packaging signal (21). Virus stocks were prepared by transient cotransfection of HeLa cells with either the pNL4-3 plasmid (1) or the C-Help vector DNA and the APO3G-expressing plasmid pcDNA-APO3G (11). Viruses were collected 48 h after transfection and purified by two rounds of sucrose gradient centrifugation. Cell lysates and concentrated virus preparations were analyzed by immunoblotting (Fig. 1A). We found that packaging of APO3G into helper virus was reduced by Ͼ3.5-fold compared to packaging into NL4-3 virus (Fig. 1B). Thus, viral RNA contributes to the specific packaging of APO3G into HIV-1 virions, consistent with data reported by Svarovskaia et al. (28).If encapsidation of APO3G and viral RNA are linked, the APO3G packaging defect observed with the helper virus ( Fig. 1A and B) should be overcome by the coexpression of packaging-competent vector-derived RNA. To test this hypothesis, several packaging vectors were constructed based on the lentiviral packaging vector pHRЈCMVGFP (15). This vector contains both HIV-1 long terminal repeats (LTRs), the 5Ј-untranslated region, 35...

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormal ciliary structure and function, impaired mucociliary clearance, and chronic middle ear, sinus, and lung disease. PCD is associated with situs inversus in approximately 50% of the patients. One proposed explanation for this relationship is that normal ciliary function plays a role in normal organ orientation, whereas organ orientation in PCD is a random event because of dysfunctional cilia in early embryonic development. Another hypothesis for the association between PCD and situs inversus is that mutated genes in PCD not only cause defective cilia, but are also linked to the control of organ laterality, such that abnormalities in this molecular pathway result in random left-right asymmetry. We report on a set of monozygotic twin women with PCD. In both patients, deficiency of the inner dynein arms was noted on ciliary ultrastructural analysis, associated with a clinical syndrome of bronchiectasis, chronic sinusitis, and middle ear disease. One of the twins has situs solitus, the other has situs inversus totalis. DNA analysis confirmed that the twins are monozygotic. This is consistent with the hypothesis that situs inversus occurring in patients with primary ciliary dyskinesia is a random but "complete" event in the fetal development of patients with PCD.

Gene transfer is an attractive option to treat the basic defect in cystic fibrosis. In a double-blind, placebo-controlled, rising-dose tolerance study in the nasal epithelium, we tested the safety and efficacy of a cationic liposome [p-ethyl-dimyristoylphosphadityl choline (EDMPC) cholesterol] complexed with an expression plasmid containing hCFTR cDNA. Eleven adult CF patients were studied in a protocol that allowed comparisons within individual subjects: vector and placebo were sprayed into alternate nostrils at intervals over 7 h. After dosing, vector-specific DNA was present in nasal lavage of all subjects for up to 10 days. There were no adverse events. The vector-treated epithelium did not exhibit a significant increase in CFTR-mediated Cl- conductance from baseline and was not different from the placebo-treated nostril: mean deltaCFTR Cl- conductance, mV +/- SEM, -1.6+/-0.4 vs -0.6+/-0.4, respectively. CFTR-mediated Cl- conductance increased toward normal during repetitive nasal potential difference measurements over the 3 days before dosing which influenced the postdosing calculations. No vector-specific mRNA was detected in the nasal epithelial scrape biopsies, although endogenous CFTR mRNA was detected in all subjects. We conclude that the lipid-DNA complex is safe, but did not produce consistent evidence of gene transfer to the nasal epithelium by physiologic or molecular measures.

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Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerizationdefective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins.Telomerase is the cellular reverse transcriptase (RT) 2 that is responsible for adding telomeric DNA repeats onto the ends of linear chromosomes (1). Found in all eukaryotes, it is a multimolecular complex of which the minimal components are a catalytic RT protein (TERT) and an integral RNA template. TERT proteins from diverse mammalian, protozoan, and yeast species appear to be related to one another evolutionarily as well as to RTs from retroviruses and cellular retroelements (2-4). Telomerase RNAs, by contrast, differ widely in length and primary sequence, and their phylogenetic relationships to one another and to retroviral genomes, if any, are unclear. Certain properties that are shared among groups of retroviral or telomerase RNAs may indicate either a common evolutionary origin or the convergent evolution of features involved in RT function. Widely divergent telomerase RNAs, for example, share certain core secondary structures that are critical for telomerase activity but have no obvious counterparts in retroviral RNAs (5, 6). The single-stranded RNA genomes of retroviruses, on the other hand, share a tendency to form stable RNA homodimers that are found in all known retroviral particles and appear to be the natural substrates for viral RTs (reviewed in Ref. 7). The assembly of these viral genomic dimers is generally initiated at a specific palindrome (i.e. a self-complementary base sequence), unique to each virus, that dimerizes readily both in vitro and within cells, and mutations in these palindromes that disrupt genomic dimer formation can markedly impair viral replication and infectivity (7). RNAs derived from at least some cellular retroelements undergo similar dimerization (4). Unexpectedly, studies from our laboratory and others have recently shown that the human telomerase RNA also dimerizes readily and that this capacity to dimerize correlates with telomerase catalytic activity in cells or cell-free extract...