Prothymosin-α Variants Elicit Anti-HIV-1 Response via TLR4 Dependent and Independent Pathways.
BACKGROUND
Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intracellular and extracellular-associated functions. Recently, we identified two new ProTα variants with potent anti-HIV activity from CD8+ T cells and cervicovaginal lavage. The first is a splice variant of the ProTα gene known as isoB and the second is the product of ProTα pseudogene 7 (p7). Similarly to iso2, the anti-HIV activity of both variants is mediated by type I IFN. Here we tested whether the immunomodulatory activity of isoB and p7 are also TLR4 dependent and determined their kinetic of release in response to HIV-1 infection.


METHODS
Type I, type III, TNF-α and IL-6 mRNA inducing activity was determined in macrophages from wild type and TLR4 knockout mice treated with recombinant ProTα variants. Supernatants from mock and HIV infected cells were analyzed by mass spectrometry in positive and negative modes for the presence of ProTα variants. In silico structural and functional analysis of ProTα variants were performed.


RESULTS
We show that both isoB and p7 upregulate IFN-β, IFN-λ1, IL-6, TNF-α and RANTES mRNAs in primary human macrophages. The potent stimulation of IFN-β by the recombinant ProTα variants in human macrophages is dependent on the TLR4 pathway, whereas the induction of TNF-α and IL-6 may also occur independently of TLR4, suggesting the interaction of ProTα variants with other signaling molecules/receptors. In silico analyses confirmed that the novel isoB and p7 variants are intrinsically disordered proteins, which lack the NLS and mass spectrometry showed release of ProTα variants within minutes post HIV-1 infection. These features are consistent with the function of ProTα variants as damage associate molecular patterns (DAMPs).


CONCLUSIONS
Our findings indicate that ProTα variants strongly inhibit viral replication mainly, but not exclusively, through TLR4 signaling and that they are released within minutes of viral infection suggesting that they may function as DAMPs.
Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
G Luca
GL
Gusella
Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
Avelino
A
Teixeira
Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
Judith
J
Aberg
Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, United States of America.|Biology Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia.|Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation.
Vladimir N
VN
Uversky
Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
Arevik
A
Mosoian
D000067531:Alarmins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D018946:Chemokine CCL5 / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D015658:HIV Infections / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000254:growth & development / Q000276:immunology; D006801:Humans; D016899:Interferon-beta / Q000235:genetics / Q000276:immunology; D007372:Interferons; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D007378:Interleukins / Q000235:genetics / Q000276:immunology; D064267:Intrinsically Disordered Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000821:virology; D051379:Mice; D018345:Mice, Knockout; D061251:Primary Cell Culture; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D016415:Sequence Alignment; D015398:Signal Transduction; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology; D000095022:Interferon Lambda
D000067531:Alarmins; D018946:Chemokine CCL5; D016899:Interferon-beta; D007372:Interferons; D015850:Interleukin-6; D007378:Interleukins; D064267:Intrinsically Disordered Proteins; D020033:Protein Isoforms; D011498:Protein Precursors; D013947:Thymosin; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; C483024:CCL5 protein, human; C000624353:interferon-lambda, human; D000095022:Interferon Lambda; C508600:IL6 protein, human; C493487:Tlr4 protein, mouse; C040782:prothymosin alpha