bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.180828; this version posted July 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Title MORT, a locus for apoptosis in the human immunodeficiency virus-type 1 antisense gene: implications for AIDS, Cancer, and Covid-19 Authors/Affiliations Linda B. Ludwig, M.D*. and Michael S. Albert, M.D. (LBL) East Aurora, NY, USA 14052 and (MSA) Department of Pathology, Buffalo Mercy Hospital, Buffalo, NY, USA 14220 Contact Corresponding author: Linda B. Ludwig, M.D.: [email protected] Abstract Apoptosis, or programmed cell death, is a fundamental requirement for life in multicellular organisms, including humans, and a mechanism to maintain homeostasis and prevent unwarranted cellular proliferations such as cancer. An antisense gene in HIV-1 (Hap) induces apoptosis in human cells. Apoptotic T cell death following HIV-1 infection leads to a compromised immune system and eventually AIDS (acquired immunodeficiency syndrome). A review of several studies that focused on long-term survivors of HIV-1 reveals that these survivors had deletion-mutations in Hap. A subset of these survivors changed course and experienced CD4+ T cell death and progression to AIDS. These individuals had virus that regained Hap gene sequence that had previously been deleted. Analysis of the changes in the genetic sequences with in vivo progression of the revertant HIV-1 virus allowed identification of a specific region in Hap we are calling MORT. MORT, in Hap RNA forms a primary microRNA-like structure. Potential human mRNAs targeted by MORT mi/siRNAs include gene/RNA sequences of X-linked inhibitor of apoptosis (XIAP), survivin, and apollon, along with many other human gene sites/RNAs. Thus MORT may be acting as an RNA antagonist to cellular IAPs thereby inducing apoptotic cell death. Surprisingly, additional potential MORT targets include viral sites in human SARS-CoV-2, including the protease, nsp5 RNA. Future uses for RNA therapy and a hypothesis for an HIV intrinsic mechanism utilizing MORT for viral anti-viral (or anti-microbial) and HIV anti-immune cell defense are proposed. Keywords HIV-AIDS, antisense gene, apoptosis, regulatory/microRNA, IAP antagonist, cancer, SARS-CoV-2 (nCoV-2019) Introduction bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.180828; this version posted July 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. To die or not to die: that is the question. The acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus type 1 (HIV-1) has long been known to be associated with the death of the CD4 + T cell (Kelleher and Zaunders, 2006)). While some controversy has been associated with the mechanism of the demise of this central cell pivotal in both B and T cell immunity, laboratory observations clearly reveal the depletion of thymocyte and CD4+ T cell populations as HIV-1 infection progresses, except in rare individuals (Barmania and Pepper, 2013; Liu, et al, 1996; Dean, et al, 1996; Samson, et al, 1996; Carrington, et al, 1999). If one puts aside the human host side of the pathogenesis equation, such as those individuals lacking a competent co- receptor (the CCR5 deletion) (Barmania and Pepper, 2013; Liu, et al, 1996; Dean, et al, 1996; Samson, et al, 1996; Carrington, et al, 1999) on the immune cells to facilitate HIV entry who thereby acquire some resistance to infection by the virus: what about the virus itself induces cell death? Is there a genetic component of HIV-1 that has reliably been shown to be associated with T cell death? The human T cell intrinsically faces the decision for division, anergy, or apoptosis at multiple stages, both in developmental processes and in ramping up (and down) a response to infection (Kolb, et al, 2017; Kerr, et al, 1972; Clarke, et al, 1993; Schwartz and Osborne, 1993; Cohen, 1993). When it comes time to die- what signal or signals must occur within the cell? More particularly, is there a signal for cell death contributed by human immunodeficiency virus? Here we propose that a specific region of the Hap antisense gene located within the HIV-1 long terminal repeat (LTR) is contributing to the programmed cell death or apoptosis observed with HIV infection (Ludwig, et al, 2006; Ludwig, 2008). Hap RNA initiates in the repeat (R) region of the HIV-1 provirus and is transcribed in the reverse direction (antisense) to the other HIV genes, overlapping the beginning portion of TAR region DNA, the TATA box, the SP1 sites and NFKb sites controlling HIV-1 sense transcription, as well as 200 nucleotides of the 3’ end of the nef gene (Figure 1B) (Ludwig, et al, 2006; Ludwig, 2008). The introduction of this antisense gene into human cells induced programmed cell death or apoptosis, as classically defined (Kerr, et al, 1972; Clarke, et al, 1993; Schwartz and Osborne, 1993; Cohen, 1993). In most experiments, performed over a decade between 1995 (the discovery of the HIV antisense initiator (HIVaINR) (U.S. patent 5,919,677) and 2005, it was a race to isolate the RNA or proteins(s) from human cells before apoptosis occurred. Because of the enhanced apoptotic cell death observed with introduction of a recombinant eukaryotic vector incorporating the Hap gene sequence (GenBank CQ767337 nt 14-367), we undertook a reanalysis of observations in two “in vivo” systems: a) a mouse model of AIDS (Lindemann, et al, 1994), with a nef/LTR transgene containing near-identical sequence to that of the Hap gene, and b) comparison of the genetic changes in the nef/LTR deleted viral species isolated in a group of HIV-1-infected individuals who changed from long term non- progressor (LTNP) status to progression to AIDS, so-called “slow progressors” bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.180828; this version posted July 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. (Gorry, et al, 2007; Deacon, et al, 1995; Churchill et al, 2004; Churchill, et al, 2006; Dyer, et al, 1999; Dyer, et al 1997; Greenway, et al 1998; Rhodes, et al, 1999; Learmont, et al 1992; Oelrichs, et al 1998; Zaunders, et al 2011; Learmont, et al, 1999). The slow progressors within the Sydney Blood Bank Cohort (SBBC) remarkably demonstrated both the “loss of function” as well as “gain of function” required for T cell death, in a natural human system prior to the institution of anti-retroviral therapy (Gorry, et al, 2007; Deacon, et al, 1995; Churchill et al, 2004; Churchill, et al, 2006; Dyer, et al, 1999; Dyer, et al 1997; Greenway, et al 1998; Rhodes, et al, 1999; Learmont, et al 1992; Oelrichs, et al 1998; Zaunders, et al 2011; Learmont, et al, 1999). The original viral deletion within these slow progressors (SP) encompassed a portion of the HIV antisense gene that was critical in the formation of an RNA primary microRNA-like secondary/tertiary structure that we are calling MORT. The HIV antisense gene sequence (GenBank CQ767337) from 270-300 shares 14 to 23 nucleotides of sequence homology with sites in or bordering 177 human genes, significant identity with seven human microRNAs (identified by BLAST on MirGeneDB), and a proposed mi/siRNA forms complementary base-pairing structures with multiple human genes important in the apoptosis pathway. X- linked inhibitor of apoptosis (XIAP), survivin, and Apollon are amongst some of the inhibitor of apoptosis proteins potentially targeted by MORT. This suggests a mechanism whereby the HIV-1 virus can induce apoptosis within the human host cell, with MORT as an antagonist to IAPs, and has potential implications for AIDS latency and applications for cancer. In yet another twist, the HIV antisense gene shares 7 sites with 11-14 nt sequence identity or complementarity with the novel coronavirus, SARS-CoV 2; implications of this will also be discussed. Results A transgenic mouse model of AIDS Lindemann, et al, 1994 described a transgenic mouse model of AIDS in which the transgene incorporated only the intact HIV-1 nef and long terminal repeat (nef/LTR) genomic region (BRU, Genbank accession number K02013), and thereby also the HIV antisense gene (Hap), under the control of mouse T-cell receptor beta chain-specific promoter and enhancer elements (Figure 1 A). Dramatic changes in the mouse lymphocyte populations were induced in the transgenic mice along with a severe immunodeficiency (Lindemann, et al, 1994). Transgenic mice demonstrating the most significant and severe changes in lymphocyte populations (B6/338L) had a single aberrant copy of the transgene integrated but also rearranged such that the mouse T-cell receptor (TCR) beta chain enhancer was downstream, instead of upstream of the mouse TCR promoter and HIV-1 nef/3’long terminal repeat sequences (Figure 1A), (Lindemann, et al, 1994). This rearrangement situated the enhancer close to the region of the HIV antisense gene in the LTR and in position to help drive antisense transcription off the intrinsic HIV-1 antisense initiator (HIVaINR) in TAR bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.180828; this version posted July 1, 2020.