The role of Aicardi-Goutières Syndrome genes in HIV-1 replication

Syndrome (AGS) as a genetic disorder in new born and young children [192] showing symptoms that resemble a congenital encephalitis caused by infection with Toxoplasma, HIV-1, CMV or Rubella (TORCH) including high levels of IFNα in the cerebrospinal fluid (CSF) and brain calcifications leading to an early progressive encephalopathy (reviewed here [193, 194]). Mutations in six different genes are associated with AGS: TREX1 [195], the three subunits of the RNase H2 complex (A,B and C) [196], SAMHD1 [197] and ADAR1 [198] (all discussed below). The mechanisms behind the disease have not been fully understood. It has been postulated that a defect in nucleic acid metabolism, more specifically accumulation of microRNAs [199], cytoplasmic single stranded DNA from endogenous

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retroelements [200] or DNA repair byproducts [201] lead to the activation of innate immune receptors such as RIG-I or DNA sensors and the subsequent up regulation of type I IFNs and other inflammatory cytokines. Surprisingly, all of the genes have been found to play a role in HIV-1 replication and innate immune sensing either restricting or facilitating HIV-1 infection (Figure 1.7).

ADAR1

Adenosine deaminase acting on RNA 1 (ADAR1) belongs to a class of enzymes that edit mRNA. The deaminated adenosine becomes an inosine and pairs with cytosine instead of thymidine introducing A to G mutations much like A3G. RNA editing changes amino acid sequences [202], splice sites [203] and introduces new miRNA binding sites [204]. ADARs have been found to be involved in innate immunity by hyper-mutating genomes of RNA viruses such as Measles [205] and vesicular stomatitis virus (VSV) and also mouse polyoma DNA virus [206, 207].

ADAR1 expression is induced by type I IFNs and it has been found to be able to modify the innate immune response by sequestering dsRNA away from PKR inhibiting the activation of PKR and acting as a suppressor of type I IFN signaling ([208, 209] and reviewed here [210, 211]). Recently it has been reported that ADAR1 mutated the HIV-1 RRE, inhibiting Rev dependent nuclear export of gag and pol transcripts and inducing mutations in Env rendering the produced particle less infectious [212]. ADAR1 knockout mice develop sever deficits in hematopoiesis and showed global upregulation of type I and type II interferon [213].

34 TREX1

The cellular 3’-to-5’ DNA repair exonuclease 1 (TREX1) [214-216] binds to the SET complex and is localized to the endoplasmic reticulum. During apoptosis induced by Granzyme A the SET complex translocates to the nucleus where TREX1 degrades nuclear DNA nicked by the DNA endonuclease NM23-H1 leading to nuclear DNA fragmentation [217]. TREX1 muations are associated with familial chilblain lupus [218], systemic lupus erythematosus (SLE) [219] and AGS [195].

Trex1-deficient mice die of circulatory failure caused by inflammatory myocarditis [220]. In cells from these mice ssDNA from endogenous retroelements accumulate [200] and lead to an IRF3 dependent type I IFN response induced by the cytosolic DNA [221]. Furthermore it has been shown that TREX1 and the SET complex inhibit autointegration of HIV-1 [222]. More recently it has been reported that during HIV-1 infection in macrophages and CD4⁺ T cells TREX1 prevents induction of type I IFN by degrading cytosolic HIV-1 cDNA. In the absence of TREX1 this cytoplasmic DNA is recognized by an unknown cytoplasmic DNA sensor signaling through TBK1, STING and IRF3 [223]. A recent publication provides evidence that this unknown DNA sensor could be the above mentioned cGAS [135].

RNASEH2

Two ribonuclease (RNase) H enzymes are present in eukaryotic cells, RNase H1 and RNase H2 [224, 225]. RNAase H2 consists of three subunits A (RH2A), the catalytic subunit, and the subunits B (RH2B) and C (RH2C), which appear to serve as an assembly platform for the complex and other binding partners. The subunit B binds PCNA, a regulator of eukaryotic DNA replication [226, 227]. RNase H1 consists of three domains, the catalytic domain, the hybrid binding domain and mitochondrial targeting sequence. Both enzymes bind RNA/DNA duplex molecules and cleave the ribonucleotide-desoxyribonucleotide bond. While RNase H2 is able to excise one single ribonucleotide [194, 228-230], RNase H1 recognizes a minimum of four ribonucleotides embedded in double-stranded DNA. The function of these enzymes is not fully understood but they are thought to play a role in DNA replication and repair. RNase H1 is necessary for mitochondrial DNA replication in mice [231].

RNase H2 is involved in removing the RNA primer used to synthesize the Okazaki fragments of the lagging strand [232] and removing ribonucleotides misincorporated by the DNA polymerase during DNA replication [233-235]. During replication of

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tandem repeats [236] or transcription of ribosomal RNA synthesis [237] RNase H2 might be needed to inhibit the formation of R loops, which occur when the nascent RNA transcript hybridizes to the unwound DNA helix. R loops lead to transcriptional termination and, in the case of tandem repeats to genomic instability. Mutations in all three subunits of RNASEH2 have been found in AGS. The G37S mutation in the subunit A shows reduced nuclease activity [229, 230, 238]. Mutations in subunits B and C mainly destabilize the complex or impaired interaction with putative binding partners. The R69W mutation in subunit C is an exception; it has nuclease activity suggesting a role of subunit C in catalysis, which means that the stability of the complex influences the catalytic activity. Rnaseh2b knockout mice are not viable and the embryos accumulated DNA damage and subsequent genomic instability [239]. In a genome-wide RNAi screen in CD4⁺ T cells RH2A was found to be promote HIV-1 replication; knock down of RH2A in a T cell line reduced HIV-1 infection 2 to 3 fold [240].

SAMHD1

The sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) was originally discovered in a dendritic cell cDNA library as a homolog of the mouse IFNγ induced gene Mg11 [241]. The SAM domain is usually involved in protein protein interactions while the HD domain was found to have a putative phosphohydrolase. Indeed recently the function of SAMHD1 was uncovered and was found to be a deoxynucleoside triphosphate triposphohyrdolase [242]. SAMHD1 is a dimer and upon binding of deoxyguanosine triphosphates (dGTP) or guanosine triphosphates (GTP) assembles into the active tetramer hydrolyzing cellular deoxynucleotide (dNTP) into nucleosides and inorganic phosphates [242-245]. The cellular function remains still unknown but it has been implicated in the regulation of IFN signaling [197] and control of dNTP pool during DNA replication [246]. SAMHD1 is highly expressed in the nucleus of myeloid cells and up regulated by type I IFNs [247, 248].

In 2011 three groups identified SAMHD1 as the cellular factor degraded by Vpx in macrophages, dendritic cells and monocytes [249, 250] and then later it was discovered that SAMHD1 also restricts HIV-1 in resting CD4⁺ T cells [251, 252].

Furthermore, CD14⁺ monocytes from AGS patients support HIV-1 replication [253].

The binding affinity of RT for dNTP is around 100 nM. In these cells the concentration

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is below that threshold for reverse transcription, terminating reverse transcription or causing incorporation of ribonucleotides into the nascent cDNA [254-256]. The accessory protein Vpx encoded by SIVMAc, SIVSM and HIV-2 is able to degrade SAMHD1 by recruiting the CUL4A E3 ligase complex via binding to DCAF1 [169, 257-259]. The removal of SAMHD1 leads to an increase of intracellular dNTP levels and reverse transcription can be completed [260, 261]. In activated CD4⁺ T cells the dNTP concentration is much higher, explaining the fact that despite of SAMHD1 expression HIV-1 is not blocked but the transmission of HIV-1 from T cells to dendritic cells is inhibited by SAMHD1 [262]. The activity of SAMHD1 not only blocks HIV-1 but a range of retroviruses including MLV and feline immunodeficiency virus (FIV) [263] and also the DNA viruses Herpes Simplex Virus 1 (HSV-1) and vaccinia virus [264]. Recent evidence suggests that SAMHD1 has an additional HIV-1 restriction activity independent of the hydrolase activity. SAMHD1 is an active 3’ to 5’

exonuclease for ssDNA and ssRNA and binds RNA and DNA with complex tertiary structures like HIV-1 gag cDNA in vitro [265]. This additional restriction capability might be regulated by phosphorylation at threonine residue 502 (T502). A phosphomimetic mutation of T592 showed no effect on the dNTP hydrolase activity but disrupted the ability of SAMHD1 to restrict HIV-1 [266, 267]. The phosphorylation status of SAMDH1 seems to be dependent on cell cycle and is dephosphorylated in arrested and type I IFN treated cells [268].