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1.
Proc Natl Acad Sci U S A ; 117(32): 19475-19486, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709741

RESUMO

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , DNA Viral/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Especificidade da Espécie , Replicação Viral
2.
J Virol ; 92(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29743357

RESUMO

Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.


Assuntos
Antígenos de Neoplasias/farmacologia , Antivirais/farmacologia , Biomarcadores Tumorais/farmacologia , Proteínas de Transporte/farmacologia , Glicoproteínas/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Proteínas de Transporte/química , Produtos do Gene env/metabolismo , Glicoproteínas/química , Infecções por HIV/virologia , Humanos , Macaca mulatta , Conformação Proteica , Homologia de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Especificidade da Espécie , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
Cell Host Microbe ; 20(4): 443-457, 2016 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-27736643

RESUMO

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection.


Assuntos
HIV-1/imunologia , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Fusão de Membrana , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo , Linfócitos T/virologia , Transporte Biológico , Linhagem Celular , DNA Viral/metabolismo , Humanos , Imunidade Inata , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia
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