Introduction

Dendritic cells play a pivotal role in the course of HIV-1 infection. They are present at the site of infection in the mucosa and their primary function is to sample the environment for pathogens and cross talk to CD4⁺ and CD8⁺ T cells as professional antigen presenting cells (APCs). They must provide three signals to activate naïve T cells. First they need to present antigen on either MHC I and II (signal 1) to be sampled by the T cell receptor (TcR). They have to be matured (surface expression of CD80 and CD86) to induce antigen specific proliferation of T cells via CD28 signaling (signal 2) and they have to provide mainly secreted IL12 to promote TH1 and CTL responses (signal 3). All the three signals have to be present to activate the adaptive immune system (reviewed here [297]). During the initial infection DCs are infected only in low numbers. This is for one due to the low level of co-receptor expression and if receptor binding and fusion takes place the activity of SAMHD1 inhibits reverse transcription and productive infection. Another reason is that most HIV-1 is probably taken up via DC-SIGN and ends up in the early endosome where it does not get degraded but can be transmitted to T cells in the draining lymphnodes [90, 298, 299]. This mode of uptake can activate the DCs and provide activation signals to T cells without presenting HIV-1 antigens activation leading to a burst of HIV-1 replication in the recipient and bystander T cells [95, 146].

The role of DC-SIGN in the transmission of HIV-1 has been questioned [300]. Even in the case of productive infection, DCs do not mature and are not able to activate T cells, they might even induce a tolerogenic T cell response [301]. Although there is one report demonstrating that DCs do mature if they are highly transduced with the help of Vpx [147], data from our group presented in this thesis and from other groups do not support that claim [113, 139, 301, 302]. The data presented in this thesis were aimed at the elucidation of how HIV-1 avoids and escapes the detection by the intrinsic and innate immunes system. DCs as one of the key players of the innate immune system were used to address these questions.

138 7.2 The Vpx-VLP gateway technology

To answer our questions we adapted a technique to genetically modify MDDCs [147, 281]. Using Vpx-VLPs to achieve MDDC transduction appears to be a powerful tool and was termed the “Vpx-VLP Gateway technology” by W.C. Greene (CSHL retroviruses meeting 2013). But MDDCs are difficult to work with and all methods for genetic manipulation have their advantages and disadvantages. Compared with the transfection of siRNA, Vpx-VLPs seem to be superior since siRNA transfection and the transfection reagents alone activated MDDCs. Not only was MX1 upregulated (Figure 2.5 B) but surface expression of CD83 was increased as well. Both markers are very robust and were never upregulated in Vpx-VLP treated cells (Figure 2.4), which is also confirmed in preliminary mass spectrometry analysis (unpublished raw data, Botinelli D.). With the Vpx-VLPs approach the use of a control vector expressing a fluorescent protein makes it easy to determine transduction rate, while with siRNA the percentage of transfected cells are usually unknown, unless a fluorescent siRNA is transfected alongside. Additionally, the use of a different color in respect to the fluorescent protein expressed by the HIV-1 or SIV reporter virus later used in an infectivity experiment (e.g. GFP vs. dsREDexpress) allows the comparison of cells transduced by the knockdown or overexpression vector and the effect on the infectivity in these cells. Furthermore, the presence of a selection cassette (e.g. puromycin) allows for a short period of selection to eliminate untransduced cells. We determined by propidium iodide staining that at a concentration of 10 µg/ml puromycin 50% of MDDCs are killed in 24 hours. This leads to a very high number of only transduced cells considering a transduction rate of 70% to 90% before selection.

One of the obvious draws backs of this method is the effect VLP treatment seems to have on monocyte to dendritic cell differentiation. Although there is no difference between VLP treated MDDCs to untreated cells in the expression of the DC marker DC-SIGN, CD1a - another DC marker - is affected in a VLP dose dependent manner (Figure 2.1 A). The fact that monocytes treated with IFNβ exhibit a similar phenotype raises the question if monocytes are able to detect HIV-1 or SIV VLPs and respond by secreting type I IFN (Figure 2.2 B)? There are some reports implicating CD16⁺ inflammatory monocytes in HIV-1 replication and the progression to AIDS [303, 304].

Even though there is a difference in VLP treated MDDC compared to untreated cells,

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VLP treated cells respond normally to PRR agonist (LPS, polydAdT or polyI:C) or type I IFN stimulation, indicating that their ability to mature is not compromised (chapter 3).

Another problem with the Vpx-VLP approach is the long-lasting effect. Once it was discovered that Vpx degrades SAMHD1 we were able to monitor its fate over the course of differentiation from monocytes to MDDCs in the presence of Vpx-VLPs.

Even 5 days after Vpx-VLP treatment SAMHD1 is not re-expressed or Vpx is still present and degrades all newly synthesized protein (chapter 5, Figure 5). This explains why we were not able to use HIV-1 in our Vpx-VLP and LPS or IFNβ treated knockdown cells. Vpx rescues HIV-1 from the antiviral state but not SIVMAC allowing us to perform the experiments with SIVMAC239-GFP. This is an important fact to keep in mind when working with MDDC manipulated using Vpx-VLPs since SAMHD1 not only inhibits a broad range of retroviruses [263] but also DNA viruses [305] and possibly other viruses as well. One way to overcome this problem was to treat the monocytes with a nucleoside mix instead of Vpx-VLPs. While this approach worked to ectopically express genes of interest (Figure 2.3, chapter 5 and Figure 6.2 G) without disrupting SAMHD1 expression and its restriction ability (chapter 5), the transduction rate was not high enough to allow expression of the knockdown vector to achieve gene knockdown (chapter 5 and data not shown). Also the nucleoside approach seems to have the same effect on CD1a expression during differentiation (Figure 2.4). Maybe with the use of a modified vector with a stronger expression of the miR30 shRNA cassette it would be possible to obtain knockdown MDDCs without having the problem of SAMHD1 degradation. This would also allow experiments addressing the function of Vpx in the context of knockdown MDDCs. Since reverse transcription seems to be delayed in monocytes (chapter 2 and 5), the best approach would be, to transduce them with a 24 hours delay to allow dNTP increase by either Vpx mediated SAMHD1 degradation or nucleoside treatment. This would ensure that reverse transcription can immediately take place after infection of the monocytes.

7.3 Vpx and SAMHD1

Our first paper (chapter 4) characterizing the rescue of HIV-1 in MDDCs in an established antiviral state was published before SAMHD1 was identified. We observed that Vpx rescues HIV-1 but not HIV-2 or SIVMAC. This led us to either invoke a type I IFN induced restriction factor, which is only restricting SIVMAC but not

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HIV-1 or which restricts both but Vpx protects only HIV-1 from its activity. In the second study we confirmed that although SAMHD1 inhibits SIVMAC in immature dendritic cells and Vpx is able to relieve that block, in LPS treated cells it is not the dNTP hydrolysis activity of SAMHD1 restricting SIVMAC. We demonstrated that by adding exogenous nucleosides to LPS treated MDDC, which partially rescued HIV-1 from the antiviral state but not SIVMAC (chapter 5, Figure 3). Also knockdown of SAMHD1 did not rescue SIVMAC from LPS treatment. This data shows that there might be a restriction factor present in mature MDDC, which is not SAMHD1 and Vpx cannot rescue SIVMAC from it. If this restriction factor also happens to act on HIV-1 but Vpx is able to rescue HIV-1 from it, this would mean that Vpx is able to degrade a second cellular factor. That would not be uncommon since other viral proteins have multiple targets as well. In a different project we are trying to identify possible targets of Vpx using a proteomic approach (Botinelli D.).

It has been known that Vpx recruits the DDB1/CUL4A E3 ligase complex by binding to the adaptor protein DCAF1 via the glutamine residue at position 76 (Q76) and the phenylalanine residue at position 80 (F80) [169, 257]. When we mutated the two residues Vpx lost the capacity to bind DCAF1 and to increase HIV-1 and SIVMAC

transduction in immature MDDC as expected. But in the presence of the antiviral state they retained the ability to rescue HIV-1. We tried to confirm these findings with a DCAF1 knockdown in MDDC but with the current knowledge that SAMHD1 is not any longer present after differentiation the question if Vpx is dependent on DCAF1 in the presence of the antiviral state is not answered by this knockdown experiment.

Nonetheless, we have confirmed that Vpx Q76A does not degrade SAMHD1 but rescues HIV-1 from the antiviral state without increasing the dNTP concentration. We could not show that Vpx Q76A is having an effect on 2-LTR circle formation, which could mean that removal of the second block depends on DCAF1 or since Vpx Q76A is not able overcome the first block - increasing the dNTP pool – the effect on 2-LTR circles might be covered by a lower amount of LTR products to start with. We tried to overcome the dNTP block by adding nucleosides in combination with Vpx Q76A but even with increased LRT products, Q76A did not further increase 2-LTR circles.

SAMHD1 tetramerization is required for its function [243], and recruitment of DCAF1 to SAMHD1 by Vpx is already enough to disrupt the enzymatic function of

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SAMHD1 [259]. I tried with no success (data not shown) to see if there is an effect of the presence of Vpx or Vpx Q76A. Vpx Q76A is able to bind SAMHD1 but cannot recruit DCAF1. The interaction between SAMHD1 and Vpx Q76A could disrupt SAMHD1 activity without degradation. It has been shown that the HIV-1 restriction by SAMHD1 is regulated by phosphorylation in a cell-cycle and type I IFN dependent manner but the dNTP hydrolase activity is constitutively active. [266, 268, 306]. This implies that SAMHD1 gains a second activity when it is dephosphorylated in resting T cells and mature myeloid cells. As said above Vpx Q76A binding to SAMHD1 without recruitment of DCAF1 could disrupt its dephosphorylation in the antiviral state. But no difference was observed on the phosphorylation status in the presence of absence of Vpx-VLPs (data not shown). Since Vpx Q76A cannot recruit DCAF1 and does not degrade SAMHD1 but still retains activity in the presence of an antiviral state, the question arises if Vpx has a second function that might not be related to the degradation of a cellular factor but might act as a facilitator. Early reports claimed that Vpr plays a role in nuclear translocation of HIV-1 in non-dividing cells [307] and that this function might be encoded by Vpx in HIV-2 and SIVSM [308]. Since the ability to degrade SAMHD1 predates the birth of Vpx [309] it might be interesting to compare different SIV Vpx and Vpr and HIV-1 Vpr, specifically Vpr from macrophage tropic HIV-1 isolates, in their ability to increase HIV-1 nuclear import. Preliminary experiments with four primary Vpr sequences did not reveal a conserved function of Vpr and Vpx in the presence of the antiviral state. Ectopic expression of these Vpr/Vpx in MDDCs using nucleoside for transduction might prove to be useful.

7.4 The role of AGS proteins in the HIV-1 replication cycle

The fact that all genes causing AGS have been reported to play a role in the replication cycle of HIV-1 and probably also of other viruses (e.g ADAR1 and measles) is not surprising. The patients suffering from AGS are born with the symptoms resembling viral encephalitis and have increased IFNα levels in the CSF.

The source of the IFNα is not known but is has been argued that it could come from aberrant micro RNA processing, cytoplasmic DNA fragments from DNA replication or the reverse transcription products from endogenous retroviruses of retrotransposons.

The increase in endogenous retroviral cDNA has been confirmed in the TREX1 mouse knockout but has not yet been shown for human cells [200]. In the case for RNASHE2 B the knockout mice show sever genomic instability and are not viable

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[239]. Just recently results obtained from a SAMHD1 knockout model have been published [310]. The absence of SAMHD1 resulted in spontaneous type I IFN production and the upregulation of ISGs. In comparison to TREX1-/- and ADAR1 -/- mice [213, 220], which show both signs of autoimmune diseases, no signs of autoimmunity were observed. Surprisingly, murine macrophages have already a 10-fold higher dNTP concentration compared to human macrophages in the presence of SAMHD1 and the dNTP levels increased to concentrations comparable to activated T cells in SAMHD1 -/- murine macrophages. The concentration of dNTP in the presence of SAMHD1 was already high enough to allow reverse transcription. But a HIV-1 vector with a RT mutation causing a lowered affinity for dNTP appeared to be restricted by SAMHD1 (reviewed here [311]).

Clearly AGS seems to be an autoimmune disease caused by the innate immune system and all AGS genes play a role in the type I IFN response. This suggests that RNase H2 is a perfect candidate to be either a HIV-1 restriction factor or facilitator.

The data already published and the data presented in this thesis show a small inhibition of HIV-1 infectivity in RNASEH2A knockdown cells, classifying RNase H2 as a HIV-1 facilitator [240]. Due to time and technical constraints the RNase H2 project could not be completed but can serve as the basis for further investigation.

There might be RNASHE2A isoforms or posttranslational modifications resulting in the two protein bands migrating at the predicted size of RNase H2A. Interestingly, Kennedy et al. have seen similar results when they compared lysates from macrophages, primary T cells and Jurkat T cells for the expression levels of RNase H2A (Figure 3. A in [255]). Jurkat cells had high levels of RNase H2A while in macrophages a RNASEH2A antibody different from the one used in this study revealed two bands as well; with the upper band showing stronger expression. One could imagine to use the antibodies for immuno-precipitate and subjecting the precipitated proteins for mass spectrometry analysis to identify the upper band.

Another approach would be to over express different reported splice forms of RNASEH2A and screen them for an effect on HIV-1 infectivity. I have shown that it is possible to overexpress the subunits of RNase H2 complex in MDDCs using nucleosides for transduction. It would be possible to express one of the AGS mutants in RNASHE2B or C (e.g. RH2C R69W) reported to destabilize the complex. The mutated subunit could poison the entire complex and act as a dominant negative

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inhibitor. RNASHE2C was expressed to similar levels as the endogenous protein and would be the perfect candidate for this type of experiment. This would allow working with MDDCs in absence of Vpx-VLPs and would increase the chances to observe an effect on the antiviral state.

7.5 TRIM5 is a PRR for the retroviral capsid

TRIM5α is a multifunctional protein of the innate immune system. It not only serves as a capsid specific restriction factor for retroviruses but it is also a signal transducer in the first wave of the type I IFN pathway, downstream of the PRR (e.g.

TLR4 ) and upstream of IRF3 (Chapter 3, Figure 3.1 and 3.2). Using knockdown experiments in THP-1 cells and MDDCs we demonstrated that the strength of the antiviral state induced by LPS or poly(I:C) (data not shown) is lower in TRIM5α knockdown cells. This can be tested by simply challenging TRIM5α knockdown cells after PRR stimulation with a type IFN sensitive virus such as VSV, NDV, SIV or HIV-1. The fold rescue of infectivity of the reporter virus then gives an estimate of the change in strength of the antiviral state. The downregulated cytokine mRNA and the decreased IFNβ and other proinflammatory cytokine secretion further supports the role of TRIM5α as a signal transducer in the type I IFN pathway.

I also tried to see an effect on DNA viruses. To that end MDDC knockdown for TRIM5α were challenged with Herpes Simplex Virus 1 (HSV-1), Vaccinia virus or Adenovirus after the antiviral state was established. No effect of the TRIM5α knockdown was observed. The explanation is that these viruses encode proteins that counteract the antiviral state: Vaccinia’s E3 protein counteracts PKR, HSV-1 is able to shut down IFNβ transcription and Adenoviruses are able to suppress STAT1 [312-314].

The two functions of TRIM5α come together in its role as a PRR for the retroviral capsid. Upon binding of a restricted capsid (N-MLV by huTRIM5α, or owl monkey TRIMCyp by HIV-1) TRIM5 is able to activate the innate immune system by generating free K63 ubiquitin chains. The same way TRIM25 activates the innate immune system after RIG-I has encountered its PAMP [291]. The dual function as signaling molecule and restriction factor is not uncommon. BST-2 for example is not only able to restrict enveloped viruses by inhibiting release of the viral particles but can also activate NF-κB [315]. If BST-2 can serve as a PRR in a similar fashion as

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TRIM5α remains to be elucidated. Recently it was discovered that another TRIM protein, TRIM21, is able to restrict adenovirus and activate the innate immune system at the same time. TRIM21 is a unique cytoplasmic Fc-receptor for IgG; it binds internalized IgG bound to adenovirus particles and initiates the disassembly of the particles. At the same time it activates the innate immune system via the formation of free K63 ubiquitin chains [316]. Interestingly, TRIM21 also serves as a signaling molecule in the cytoplasmic DNA sensing pathway [317].

7.6 Final thoughts

TRIM5α knockdown MDDCs produced 13 times less IL12b after LPS stimulation.

IL12 is the main cytokine directing TH1 development [318]. IL12 production by dendritic cells upon recognition of retroviral capsid would be necessary to induce a robust T cell response able to control HIV-1. The last attempt for a HIV-1 vaccine showed some promising results. The probability of an HIV-1 infection was 26% lower in vaccinated subjects but there was no difference in viral load or CD4⁺ T cell count in vaccinated individuals after HIV-1 infection [319]. This means that we still do not know what kind of immune response needs to be elicited to either block the initial infection or to control the systemic infection. As mentioned above one of the problems is the ability of HIV-1 to hide from the innate immune system preventing activation of dendritic cells. Our data shows that TRIM5a has the ability to recognize the retroviral capsid, activate dendritic cells and induce the production of IL12 to possibly promote a TH1 response.

Although some secreted IFNβ from MDDCs challenged with N-MLV VLPs can be detected (Figure 3.3 B), we never observed full activation of DCs. This second signal would be necessary for T cell activation. What is missing is the induction of IFNβ after detection of reverse transcription products. As previously mentioned, pDCs are able to detect HIV-1 genomic RNA via TLR7 and 9 and produce IFNβ but conventional DCs lack this ability [143]. One explanation lies in the presence of TREX1. As mentioned before, TREX1 facilitates HIV-1 infection by degrading cytoplasmic DNA, which would otherwise be detected by cGAS and induce an IFNβ

Although some secreted IFNβ from MDDCs challenged with N-MLV VLPs can be detected (Figure 3.3 B), we never observed full activation of DCs. This second signal would be necessary for T cell activation. What is missing is the induction of IFNβ after detection of reverse transcription products. As previously mentioned, pDCs are able to detect HIV-1 genomic RNA via TLR7 and 9 and produce IFNβ but conventional DCs lack this ability [143]. One explanation lies in the presence of TREX1. As mentioned before, TREX1 facilitates HIV-1 infection by degrading cytoplasmic DNA, which would otherwise be detected by cGAS and induce an IFNβ