Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA

Origin and functional role of antisense transcription in endogenous and exogenous retroviruses

Abstract Most proteins expressed by endogenous and exogenous retroviruses are encoded in the sense (positive) strand of the genome and are under the control of regulatory elements within the 5’ long terminal repeat (LTR). A number of retroviral genomes also encode genes in the antisense (negative) strand and their expression is under the control of negative sense promoters within the 3’ LTR. In the case of the Human T-cell Lymphotropic Virus 1 (HTLV-1), the antisense protein HBZ has been shown to play a critical role in the virus lifecycle and in the pathogenic process, while the function of the Human Immunodeficiency Virus 1 (HIV-1) antisense protein ASP remains unknown. However, the expression of 3’ LTR-driven antisense transcripts is not always demonstrably associated with the presence of an antisense open reading frame encoding a viral protein. Moreover, even in the case of retroviruses that do express an antisense protein, such as HTLV-1 and the pandemic strains of HIV-1, the 3’ LTR-driven antisense transcript shows both protein-coding and noncoding activities. Indeed, the ability to express antisense transcripts appears to be phylogenetically more widespread among endogenous and exogenous retroviruses than the presence of a functional antisense open reading frame within these transcripts. This suggests that retroviral antisense transcripts may have originated as noncoding molecules with regulatory activity that in some cases later acquired protein-coding function. Here, we will review examples of endogenous and exogenous retroviral antisense transcripts, and the ways through which they benefit viral persistence in the host

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus

The HIV-1 Antisense Gene ASP: The New Kid on the Block

Viruses have developed incredibly creative ways of making a virtue out of necessity, including taking full advantage of their small genomes. Indeed, viruses often encode multiple proteins within the same genomic region by using two or more reading frames in both orientations through a process called overprinting. Complex retroviruses provide compelling examples of that. The human immunodeficiency virus type 1 (HIV-1) genome expresses sixteen proteins from nine genes that are encoded in the three positive-sense reading frames. In addition, the genome of some HIV-1 strains contains a tenth gene in one of the negative-sense reading frames. The so-called Antisense Protein (ASP) gene overlaps the HIV-1 Rev Response Element (RRE) and the envelope glycoprotein gene, and encodes a highly hydrophobic protein of ~190 amino acids. Despite being identified over thirty years ago, relatively few studies have investigated the role that ASP may play in the virus lifecycle, and its expression in vivo is still questioned. Here we review the current knowledge about ASP, and we discuss some of the many unanswered questions