APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells.

While the ability of APOBEC3G to reduce the replication of a range of exogenous retroviruses is now well established, recent evidence has suggested that APOBEC3G can also inhibit the replication of endogenous retrotransposons that bear long terminal repeats. Here, we extend this earlier work by showing that two other members of the human APOBEC3 protein family, APOBEC3B and APOBEC3A, can reduce retrotransposition by the intracisternal A-particle (IAP) retrotransposon in human cells by 20-fold to up to 100-fold, respectively. This compares to an approximately 4-fold inhibition in IAP retrotransposition induced by APOBEC3G. While both APOBEC3G and APOBEC3B specifically interact with the IAP Gag protein in co-expressing cells, and induce extensive editing of IAP reverse transcripts, APOBEC3A fails to package detectably into IAP virus-like particles and does not edit IAP reverse transcripts. These data, which identify human APOBEC3A as a highly potent inhibitor of LTR-retrotransposon function, are the first to ascribe a biological activity to APOBEC3A. Moreover, these results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBEC3G and APOBEC3B.

Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide.

Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability.

A potent anti-HIV immunotoxin blocks spreading infection by primary HIV type 1 isolates in multiple cell types.

Although several immunotoxins that selectively kill HIV-1-infected cells have been described, their clinical utility is limited by low potency against spreading viral infection. We show here that changing the carboxyterminal sequence of an anti-HIV-1 envelope immunotoxin to the consensus endoplasmic reticulum retention sequence KDEL substantially improves its ability to block infection of peripheral blood mononuclear cells by primary HIV-1 isolates without increasing nonspecific toxicity. Polychromatic flow cytometry of peripheral blood mononuclear cells (PBMC) infected with an HIV-1-GFP reporter virus demonstrated that the improved immunotoxin is active against a variety of primary cell types including memory T cells, NK-T cells, and monocyte/macrophages. The subnanomolar potency of this agent suggests that it could be clinically useful either as an adjuvant to highly active antiretroviral therapy (HAART) in drug-resistant patients or to reduce the reservoir of latently infected cells that is implicated in HIV-1 persistence.

Genetic and functional analysis of single nucleotide polymorphisms in the beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2).

The beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2) is a logical candidate for influencing smoking behavior and nicotine dependence. We discovered six single nucleotide polymorphisms (SNPs) in the CHRNB2 gene by surveying 15.4 kb of genomic sequence including a previously undescribed 3' untranslated region that extends 4.0 kb downstream of the coding region. One of the SNPs causes an amino acid substitution in exon 5, one occurs in the promoter region, one changes an intronic base, and three occur in the 3' untranslated region. The ethnically dependent allele frequencies and the marker-to-marker linkage disequilibrium patterns of five of these polymorphisms were determined. The SNPs were assayed in 743 individuals for whom information on smoking history and lifelong nicotine dependence was available. No significant associations of the individual markers or their haplotypes to smoking behavior or level of nicotine dependence were found.