Quasispecies as a matter of fact: Viruses and beyond

We review the origins of the quasispecies concept and its relevance for RNA virus evolution, viral pathogenesis and antiviral treatment strategies. We emphasize a critical point of quasispecies that refers to genome collectivities as the unit of selection, and establish parallels between RNA viruses and some cellular systems such as bacteria and tumor cells. We refer also to tantalizing new observations that suggest quasispecies behavior in prions, perhaps as a result of the same quantum-mechanical indeterminations that underlie protein conformation and error-prone replication in genetic systems. If substantiated, these observations with prions could lead to new research on the structure–function relationship of non-nucleic acid biological molecules

A Matter of Life or Death: Productively Infected and Bystander CD4 T Cells in Early HIV Infection

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24+) cells and bystander (p24-) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24+ cells had longer telomeres compared to p24- cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24+ cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection

Contributions to the Mathematical Systems Medicine of Antimicrobial Therapy and Genotype-Phenotype Inference.

The following summary of my publications describes the main ideas in the corresponding research articles and clarfifies my contribution in multi-author publications. I decided to apply for habilitation according to x2.I.1.(c) of the Habilitationsordnung (this path is usually referred as Kumulative Habilitation"). I selected 13 first- or last author publications for this habilitation that concern contributions to the mathematical systems medicine of antiviral therapy [tMH10, tMS+11, FtK+11, tMMS12, DSt12, DWSt15, Dt16, DSt16, DDKt18, DSD+19, DDKt19], as well as inference of genotype-phenotype associations [SDH+15, SSJ+18]. The selected publications represent my major contributions in this research eld since submitting my doctoral thesis in September 2009

Alcohol as a catalyst for hiv-associated neuroinflammation and tbi-induced iron toxicity

Alcohol has long been considered an exacerbator of diseases, disorders, and injuries as well as many of the accompanying symptoms. As an alternative approach, this dissertation explores alcohol as a catalyst for two different human disease conditions, human immunodeficiency virus (HIV)-associated neuroinflammation and traumatic brain injury (TBI)-induced iron toxicity. In HIV-1 infection, this dissertation presents a novel anti-viral drug, called Drug-S, for a possible inhibition and treatment of HIV-1 disease progression. The first aim explores the influence of alcohol with HIV-associated neuroinflammation on macrophage migration across an in vitro model of the blood brain barrier. There is a gap in knowledge on the effects of low dose alcohol under HIV-associated injury in people living with HIV-1 who have achieved viral suppression. The model, consisting of a quad-cultivation of neuroimmune cells including endothelial, astrocyte, macrophage, and neuron cells, is challenged with low dose (10 mM) alcohol and the viral protein trans-activator of transcription (TAT). It was then observed for changes to barrier integrity and neuronal injury upon macrophage migration. Results show that combined alcohol and viral injuries significantly increases migration even under the clinically lowest concentrations of alcohol. The cause of enhanced macrophage migration and related neurotoxicity is implicated to alcohol-induced nitric oxide production by endothelial cells and TAT\u27s chemoattractant properties. The second aim analyzes a compound called Drug-S as a possible therapeutic for inhibiting HIV-1 replication and HIV-1 disease progression. Although the combination of highly active antiretroviral therapy can remarkably control HIV-1, it is not a cure. Current therapy is unable to eliminate persistent HIV-1 contained in latent reservoirs in the central nervous system and to prevent rebound viral replication and resurgence when treatment is withdrawn. Treating HIV-1 infected macrophage with Drug-S shows inhibition of infection and persistence at a low concentration without causing any toxicity to neuroimmune cells. Results suggest that Drug-S may have a direct effect on viral structure, prevent rebounding of HIV-1 infection, and arrest progression into acquired immunodeficiency syndrome. The third aim explores the role of low level of alcohol use in TBI-induced hemolytic iron management. As hemorrhage is a major component of TBI, the accumulated red blood cells in the tissue layers undergo hemolysis and release free iron into the central nervous system. As a secondary stressor, prior alcohol consumption can increase iron aggregation and alter its management. The effects of alcohol on TBI- induced iron toxicity is explored in an in vivo model of chronic alcohol exposure subjected to fluid percussion injury. Results show that alcohol increases the iron overload and alters iron management following injury by changing the expression profile of the iron regulatory proteins lipocalin 2, heme oxygenase 1, ferritin light chain, and hemosiderin. Accompanying these results, it was also found that microglia can similarly play a significant role in iron management by phagocytosing red blood cells and retaining iron. Overall, the results of this dissertation demonstrate the pervasive impact of alcohol use in neuropathophysiology arising from HIV protein TAT toxicity or TBI-induced iron toxicity. In addition, the newly discovered DrugS can be an effective antiviral drug for a possible HIV/AIDS disease prevention and progression

A Matter of Life or Death: Productively Infected and Bystander CD4 T Cells in Early HIV Infection

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24+) cells and bystander (p24-) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24+ cells had longer telomeres compared to p24- cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24+ cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection

Control and prevention of infectious diseases from a One Health perspective

The ongoing COVID-19 pandemic has caught the attention of the global community and rekindled the debate about our ability to prevent and manage outbreaks, epidemics, and pandemics. Many alternatives are suggested to address these urgent issues. Some of them are quite interesting, but with little practical application in the short or medium term. To realistically control infectious diseases, human, animal, and environmental factors need to be considered together, based on the One Health perspective. In this article, we highlight the most effective initiatives for the control and prevention of infectious diseases: vaccination; environmental sanitation; vector control; social programs that encourage a reduction in the population growth; control of urbanization; safe sex stimulation; testing; treatment of sexually and vertically transmitted infections; promotion of personal hygiene practices; food safety and proper nutrition; reduction of the human contact with wildlife and livestock; reduction of social inequalities; infectious disease surveillance; and biodiversity preservation. Subsequently, this article highlights the impacts of human genetics on susceptibility to infections and disease progression, using the SARS-CoV-2 infection as a study model. Finally, actions focused on mitigation of outbreaks and epidemics and the importance of conservation of ecosystems and translational ecology as public health strategies are also discussed

Structural determinants of murine leukemia virus reverse transcriptase that are important for template switching, fidelity, and drug resistance

Retroviruses exhibit high mutation rates. High mutation and recombination rates increase variation within a viral population and result in production of drug-resistant mutants and/or mutants that can escape a host immune response. Mutations are introduced into the viral genome by error-prone reverse transcriptase (RT), a virally encoded enzyme that converts single-stranded viral RNA into double-stranded DNA. In the current study we are trying to understand what structural determinants of RTs are important for fidelity, frequency of template switching, and drug-resistance. First, we developed an in vivo assay and performed mutational analysis of manne leukemia virus (MLV) RT to identify structural elements important for template switching. Based on obtained results, we proposed a dynamic copy-choice model in which both the rate of DNA polymerization and the rate of degradation of the RNA template influence the frequency of RT template switching. Second, we employed a previously described in vivo fidelity assay to determine whether a minor groove binding helix of the thumb domain and primer grip of MLV RT are important for in vivo fidelity of reverse transcription. Because the thumb domain of MLV RT has not been crystallized, we utilized homology alignment and molecular modeling to identify the minor groove binding helix of the thumb domain of MLV RT. Mutations in the minor groove binding helix residues R301 and F309 decreased RT fidelity by up to 2.8-fold, suggesting that this region plays an important role in accuracy of DNA synthesis. Finally, we attempted to elucidate a mechanism of drug-resistance to the antiretroviral nucleoside analog 2\u27,3\u27-dideoxy-3 \u27-thiacytidine (3TC), an inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) RT. We tested our hypothesis that a valine residue at the 223 position in YVDD motif of the MLV RT leads to a natural high level of resistance of MLV to 3TC in a manner similar to that proposed for the YVDD mutant of HIV-1 RT. The results indicated that the wild-type, V223M, V2231, V223A, and V223S mutants of MLV RT were all highly resistant to 3TC, suggesting that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC

Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV

Role of HIV-1 and Bacteremia Co-infection in Promoting Inflammatory Mediator production in Kenyan Children with Severe Malarial Anemia

Severe anemia is the primary outcome of childhood malaria in holoendemic malaria transmission regions such as western Kenya. HIV-1 and bacteremia are equally important diseases in Kenyan children. Anemia is also the hallmark trait of pediatric HIV-1 infection, and despite our previous reports of exacerbated anemia in malaria/HIV-1 co-infected childrena, we also observed significantly lower parasitemias without worsening anemia in malaria/bacteremia co-infected children. Abundant production of pro-inflammatory cytokines is known to adversely affect erythropoiesis and is common to malaria, HIV-1, and bacteremia. As such, we performed a comprehensive bead-based 25-plex Multiplex assay to identify cytokines patterns and profiles associated with negative and positive hematolgic outcomes in co-infected children. Children in Aims 1 and 2 infected with P. falciparum malaria (Pf[+], aged 3-36 mos., n=542) were stratified into three groups: HIV-1 negative (HIV-1[-]/Pf[+]); HIV-1 exposed (HIV-1[exp]/Pf[+]); and HIV-1 infected (HIV-1[+]/Pf[+]). In Aim 3, malaria and bacteremia co-infected children (n=192) were divided into three categories: malaria alone, Pf[+]; Gram negative bacteremia/malaria co-infected, G[-]/Pf[+]; and Gram positive bacteremia/malaria co-infected, G[+]/Pf[+]. Univariate, correlational, and hierarchical regression analyses were used to determine differences among the groups and to define predictors of worsening anemia. Aim 1 analyses revealed HIV-1[+]/Pf[+] children had significantly more malarial pigment-containing neutrophils (PCN), monocytosis, increased severe anemia (Hb<6.0g/dL), and ~10-fold greater mortality. Hierarchical multiple regression revealed that worsening anemia was associated with elevated pigment-containing monocytes, younger age, and increasing HIV-1 status (HIV-1[-]→HIV-1[exp]→HIV-1[+]). Aim 2, addressing the inflammatory milieu, demonstrated that exacerbated anemia was associated with inflammatory mediator (IM) dysregulation, but not parasitemic or erythropoietic indices. A principal component analysis revealed that IL-12 was the most influential variable on Hb levels in HIV-1[+]/Pf[+] children, while the IL-1β:IL-10 ratio was most influenced by PCN. In Aim 3, both bacteremia co-infected groups had lower parasitemia compared to the Pf[+] group. A multiple mediation model examining IMs responsible for decreased parasitemia in the bacteremia co-infected groups identified IL-4, IL-10, IL-12, and IFN-γ as the key molecules in decreasing parasitemia. Thus, malaria/HIV-1 co-infection is defined by significantly enhanced anemia that is associated with unique IM profiles known to exacerbate anemia, while enhanced immune activation in malaria/bacteremia co-infected children appears to promote reduced parasitemia without adversely affecting anemia outcomes. By defining the inflammatory milieu associated with severe anemia, therapies can be developed to mitigate detrimental immune responses, thereby lessening the pediatric public health burden in western Kenya

The role of Gag in HIV-1 DNA synthesis and sensitivity to reverse transcriptase inhibitor drugs

We hypothesise that HIV-1 DNA synthesis occurs inside intact viral capsid (CA) cores. We propose that dNTPs are transported into the CA via an electrostatic channel, formed by six positively charged arginines in the centre of CA hexamers. Here, we consider whether reverse transcriptase inhibitor (RTI) sensitivity is altered when the nature of the channel is changed, either by Gag mutation or exchange with Gag from a non-pandemic HIV isolate with a different structure. There are two classes of RTIs: nucleoside/nucleotide based (NRTI) and non-nucleoside inhibitors (NNRTI). We hypothesised that negatively charged NRTIs would recruit to CA hexamers, to be transported into cores. However, NNRTIs are uncharged, yet potently inhibit DNA synthesis, suggesting that NNRTIs enter cores by diffusion or inhibit after uncoating. We tested HIV-1 vector sensitivity to RTIs, either bearing lab adapted M-group, transmitted founder, O-group or mutant Gag sequences. Viral inhibition was measured by comparing IC50 and IC90 values in a range of cell lines. Our data shows that some differences in Gag demonstrate a cell type-dependent effect on viral sensitivity to RTIs. We also tested the stage of RTI inhibition, measuring early and late–reverse transcription (RT) products of HIV-1 (M) and HIV-1 (O) virus in the presence of inhibitors. Our data show that both HIV-1 (M) and (O) vectors are inhibited after 2nd DNA strand transfer. We determined that a small number of vDNA strands are required to infect a U87 cell, which increases in the presence of RTIs or on R18G mutation. We conclude that differences in Gag have some small cell type-dependent effects on RTI sensitivity. We hypothesise this may be due to differences in the timing of CA uncoating between cell types, supported by our finding that all RTIs tested inhibit RT predominantly after 2nd strand transfer