Review The HIV-1 Antisense Gene ASP: The New Kid on the Block Zahra Gholizadeh, Mohd. Shameel Iqbal, Rui Li and Fabio Romerio * Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; [email protected] (Z.G.); [email protected] (M.S.I.); [email protected] (R.L.) * Correspondence: [email protected] Abstract: 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. Keywords: HIV-1; natural antisense transcription; antisense protein (ASP); overprinting; replication; pathogenesis; spread Citation: Gholizadeh, Z.; Iqbal, M.S.; Li, R.; Romerio, F. The HIV-1 Antisense Gene ASP: The New Kid 1. Introduction: De Novo Creation of Genes on the Block. Vaccines 2021, 9, 513. In the majority of cases, new genes are created by transfer of existing genetic mate- https://doi.org/ rial [1]. This can occur in various forms: exon shuffling, gene duplication, retroposition, 10.3390/vaccines9050513 lateral gene transfer, and gene fusion or fission [1]. However, in rare cases, genes can also be created de novo. This mechanism was thought to be rare [2], but recent studies show that it Academic Editor: Mudit Tyagi occurs frequently. De novo gene creation can occur in intergenic regions and introns [3,4], but also in genomic regions that already contain a protein-coding gene—a process called Received: 20 April 2021 ‘overprinting’ (Figure1). In this case, a genomic region with an existing coding sequence in Accepted: 13 May 2021 one of the six reading frames (Figure1A) undergoes point mutations in one of the other Published: 17 May 2021 five reading frames that generate a new start codon (Figure1B) and/or remove stop codons (Figure1C), giving rise to a second coding sequence. The resulting genomic region contains Publisher’s Note: MDPI stays neutral two overlapping open reading frames (ORFs): the ancestral or ‘overprinted’ gene, and the with regard to jurisdictional claims in novel or ‘overprinting’ gene [5]. For most gene pairs, the identification of the ancestral published maps and institutional affil- and the de novo gene can be determined with high accuracy because the former presents a iations. much wider phylogenetic distribution than the latter [6]. Indeed, proteins that are created de novo by overprinting do not have homologs in other organisms and are at times referred to as taxonomically restricted or ‘ORFans’ [7,8]. However, it is also possible that de novo proteins may be members of a family of proteins that have diverged beyond recognition or Copyright: © 2021 by the authors. that were lost [9]. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Vaccines 2021, 9, 513. https://doi.org/10.3390/vaccines9050513 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, x 2 of 15 Vaccines 2021, 9, 513 2 of 15 A. +3 +2 Ancestral gene +1 -1 -2 -3 Creation of start in +3 reading frame Removal of stop in -2 reading frame Novel/overprinting gene B.+3 C. +3 +2 +2 Ancestral/overprinted gene Ancestral/overprinted gene +1 +1 -1 -1 Novel/overprinting gene -2 -2 -3 -3 Figure 1. De novo creation of genes by overprinting. Typically, genomic regions contain a single gene encoding a protein Figureproduct 1. inDe one novo of creation the 6 reading of genes frames by overprinting. (for example, Typically, reading framegenomi +1c regions in panel contain (A). The a single same genomicgene encoding region a doesprotein not productencode anyin one protein of the in 6 the reading other 5frames reading (for frames, example, either reading because frame of absence +1 in panel of start (Acodons). 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Whena new thisstart occurs, codon (as the in original reading gene frame is called +3 in panel ‘ancestral’ (B) or or by ‘overprinted’, eliminating a while stop codon the new (as one in reading is called frame‘novel’ −2 or in ‘overprinting’. panel (C). When In rare this cases,occurs, both the events original can gene occur is called over time ‘anc (asestral’ in the or case‘overprinted’, of HTLV-1 while and HIV-1), the new thus one giving is called rise ‘novel’ or ‘overprinting’. In rare cases, both events can occur over time (as in the case of HTLV-1 and HIV-1), thus giving to genomic regions that contain three overlapping protein-coding genes. rise to genomic regions that contain three overlapping protein-coding genes. 2.2. Overprinting Overprinting in in Viral Viral Genomes Genomes OverprintingOverprinting has has been been documented documented in both prokaryotic in both prokaryotic and eukaryotic and genomes eukaryotic [10– 17].genomes However, [10– the17]. incidence However, of the overlapping incidence ofge overlappingnes in these organisms genes in these is relatively organisms low is [18– rela- tively low [18–20]. On the other hand, overprinting is quite frequent in viral genomes [21, 20]. On the other hand, overprinting is quite frequent in viral genomes [21,22]. Indeed, the 22]. Indeed, the first evidence of overlapping genes came from the bacteriophage FX174 [23], first evidence of overlapping genes came from the bacteriophage ΦX174 [23], then fol- then followed by many examples in several eukaryotic viruses. Two theories have been lowed by many examples in several eukaryotic viruses. Two theories have been proposed proposed to explain the high abundance of overlapping genes in viral genomes [24]. The to explain the high abundance of overlapping genes in viral genomes [24]. The gene-com- gene-compression theory states that error-prone viral polymerases and biophysical con- pression theory states that error-prone viral polymerases and biophysical constraints im- straints imposed by the viral capsid drive the creation of overlapping genes that allow posed by the viral capsid drive the creation of overlapping genes that allow maximization maximization of the amount of information that can be encoded in small genomes [25]. The of the amount of information that can be encoded in small genomes [25]. The gene-novelty gene-novelty theory asserts that de novo creation of genes is driven by selective pressure, theory asserts that de novo creation of genes is driven by selective pressure, and gives rise and gives rise to gene products that provide a selective advantage to the virus, and thus to gene products that provide a selective advantage to the virus, and thus become fixed become fixed in the population [5]. in the Thepopulation high abundance [5]. of overlapping genes in viral genomes has allowed the use of statisticalThe high andabundance computational of overlapping methods genes to investigate in viral genomes the composition has allowed bias, the structural use of statisticalfeatures, and evolution, computational and potential methods function to investigate of overlapping the composition gene pairs bias, and structural de novo fea- pro- tures,teins evolution, [5,24,26,27 ].and Sequence potential composition function of overlapping varies greatly gene between pairs and overlapping de novo proteins and non- [5,24,26,27].overlapping Sequence genes, as composition well as between varies ancestral greatly and between novel overlapping genes in overlapping and non-overlap- pairs [27]. pingPavesi genes, et al. as showedwell as between that, in theancestral case of and overlapping novel genes pairs, in overlappi the codonng usagepairs [27]. of ancestral Pavesi etgenes al. showed is more that, similar in the to case the restof overlapping of the genome pairs, than the that codon of novel usage genes of ancestral [26]. 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