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Rabbits as a model to study HIV-1 infection and the search for new innate immunity players in resting CD4 T cells
Rabbits as a model to study HIV-1 infection and the search for new innate immunity players in resting CD4 T cells
Once infected with human immunodeficiency virus (HIV), currently available pharmacotherapies can only partly control, but not cure infection. Thus, there remains an urgent need for more potent and conceptually novel antiviral therapeutics, including the development of prophylactic and therapeutic HIV vaccines. To develop such novel treatment strategies, the use of animal models is critical to study virus replication and disease progression in vivo. On the other side, understanding how innate immunity works in primary human HIV target cells is important to find novel measures to inhibit or even eradicate HIV. Furthermore, knowledge gained from innate immunity studies in human cells can be transferred to animal model development. Here, we focus on both establishing rabbits as a suitable candidate to study HIV pathogenesis and identifying SAM domain and HD domain-containing protein 1 (SAMHD1) and a currently unknown protein as important cellular factors inhibiting HIV replication in primary resting T cells. Cells from New Zealand white rabbits display a remarkable HIV susceptibility ex vivo as they express only three blocks to full-length HIV replication. Deficits at the level of entry and reverse transcription could be overcome by transient expression of human CD4/CCR5 on primary rabbit macrophages and by using a HIV/ simian immunodeficiency virus (SIV) capsid chimera to avoid recognition by rabbit tripartite motif-containing protein 5 (TRIM5). The nature of the third barrier, causing a HIV infectivity defect in primary rabbit macrophages, remains elusive. As the phenotype resembles the antiviral activity of serine incorporator proteins 3/5 (SERINC3/5), we analyzed SERINC3/5 orthologs from mouse, rat and rabbit, and compared them to the human counterparts. We found that all orthologs are highly conserved at amino acid level. In the absence of viral antagonists, all rodent and lagomorph SERINC3 and SERINC5 orthologs displayed anti-HIV activity comparable to the human orthologs, generally with lower restriction activities for SERINC3 than for SERINC5. Interestingly, HIV Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia (EIAV) S2 proteins counteracted the antiviral activity of all SERINC3/5 orthologs with comparable efficiencies. Thus, our results demonstrate that the antiviral activity of SERINC proteins is conserved also in rodents and rabbits, and can be overcome by all three thus far identified viral antagonists. These findings indicate that SERINC3/5 restrictions do not pose a significant barrier for the development of immunocompetent animal models for HIV-1 infection. Resting CD4 T cells are one of the major target cells for HIV. Since two decades it was known that resting CD4 T cells are highly resistant to productive infection by inhibiting early reverse transcription of incoming viral genomes, but its underlying nature remained elusive. Here, we identified the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 as a major restriction factor acting also in resting CD4 T cells. SAMHD1 reduces intracellular dNTP pools, which are a major substrate for the reverse transcription of HIV-1 RNA to cDNA. This restriction is overcome by HIV-1 or HIV-2 virions into which viral protein X (Vpx) is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx from the SIVmac (rhesus macaque)/HIV-2 lineage mediates proteasomal degradation of SAMHD1, which leads to the elevation of intracellular deoxynucleotide pools and successful infection of Vpx-carrying HIV. Subsequently, we found that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages. Surprisingly, these Vpx proteins did not induce SAMHD1 degradation, dNTP pool elevation, or change SAMHD1 phosphorylation. We mimicked enhancement of early post entry steps in a Vpx rcm/mnd-2-like fashion by generating single amino acid changes in the SAMHD1-degrading Vpx mac239 protein. In addition, SIVmac239 Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. Thus, our results indicate that Vpx can also counteract an additional block at the level of reverse transcription that acts independently of the SAMHD1-mediated restriction and is specific to resting CD4 T cells. Summarizing, identification, characterization and surmounting of barriers to HIV replication will increase our knowledge on HIV innate immunity and help to build an immunocompetent small animal model to HIV infection.
HIV; innate immunity; animal model
Baldauf, Hanna-Mari
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Baldauf, Hanna-Mari (2018): Rabbits as a model to study HIV-1 infection and the search for new innate immunity players in resting CD4 T cells. Habilitationsschrift, LMU München: Faculty of Medicine
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Abstract

Once infected with human immunodeficiency virus (HIV), currently available pharmacotherapies can only partly control, but not cure infection. Thus, there remains an urgent need for more potent and conceptually novel antiviral therapeutics, including the development of prophylactic and therapeutic HIV vaccines. To develop such novel treatment strategies, the use of animal models is critical to study virus replication and disease progression in vivo. On the other side, understanding how innate immunity works in primary human HIV target cells is important to find novel measures to inhibit or even eradicate HIV. Furthermore, knowledge gained from innate immunity studies in human cells can be transferred to animal model development. Here, we focus on both establishing rabbits as a suitable candidate to study HIV pathogenesis and identifying SAM domain and HD domain-containing protein 1 (SAMHD1) and a currently unknown protein as important cellular factors inhibiting HIV replication in primary resting T cells. Cells from New Zealand white rabbits display a remarkable HIV susceptibility ex vivo as they express only three blocks to full-length HIV replication. Deficits at the level of entry and reverse transcription could be overcome by transient expression of human CD4/CCR5 on primary rabbit macrophages and by using a HIV/ simian immunodeficiency virus (SIV) capsid chimera to avoid recognition by rabbit tripartite motif-containing protein 5 (TRIM5). The nature of the third barrier, causing a HIV infectivity defect in primary rabbit macrophages, remains elusive. As the phenotype resembles the antiviral activity of serine incorporator proteins 3/5 (SERINC3/5), we analyzed SERINC3/5 orthologs from mouse, rat and rabbit, and compared them to the human counterparts. We found that all orthologs are highly conserved at amino acid level. In the absence of viral antagonists, all rodent and lagomorph SERINC3 and SERINC5 orthologs displayed anti-HIV activity comparable to the human orthologs, generally with lower restriction activities for SERINC3 than for SERINC5. Interestingly, HIV Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia (EIAV) S2 proteins counteracted the antiviral activity of all SERINC3/5 orthologs with comparable efficiencies. Thus, our results demonstrate that the antiviral activity of SERINC proteins is conserved also in rodents and rabbits, and can be overcome by all three thus far identified viral antagonists. These findings indicate that SERINC3/5 restrictions do not pose a significant barrier for the development of immunocompetent animal models for HIV-1 infection. Resting CD4 T cells are one of the major target cells for HIV. Since two decades it was known that resting CD4 T cells are highly resistant to productive infection by inhibiting early reverse transcription of incoming viral genomes, but its underlying nature remained elusive. Here, we identified the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 as a major restriction factor acting also in resting CD4 T cells. SAMHD1 reduces intracellular dNTP pools, which are a major substrate for the reverse transcription of HIV-1 RNA to cDNA. This restriction is overcome by HIV-1 or HIV-2 virions into which viral protein X (Vpx) is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx from the SIVmac (rhesus macaque)/HIV-2 lineage mediates proteasomal degradation of SAMHD1, which leads to the elevation of intracellular deoxynucleotide pools and successful infection of Vpx-carrying HIV. Subsequently, we found that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages. Surprisingly, these Vpx proteins did not induce SAMHD1 degradation, dNTP pool elevation, or change SAMHD1 phosphorylation. We mimicked enhancement of early post entry steps in a Vpx rcm/mnd-2-like fashion by generating single amino acid changes in the SAMHD1-degrading Vpx mac239 protein. In addition, SIVmac239 Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. Thus, our results indicate that Vpx can also counteract an additional block at the level of reverse transcription that acts independently of the SAMHD1-mediated restriction and is specific to resting CD4 T cells. Summarizing, identification, characterization and surmounting of barriers to HIV replication will increase our knowledge on HIV innate immunity and help to build an immunocompetent small animal model to HIV infection.