Chapter 7: General discussion
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 response via STING signaling [135, 223]. Under normal circumstances TREX1 probably prevents overstimulation of the innate immune system by endogenous retroviral elements and DNA replication [220, 320]. TREX1 is one of the two
145
nucleases of the SET complex, which fragments DNA after activation by granzyme A induced apoptosis [217].
Interestingly, knockdown of SET complex members can protect HIV-1 from autointegration, a process where HIV-1 IN mistakenly integrates into itself instead of the host genome [222]. It was also shown that uracilated HIV-1 cDNA is less likely to succumb to autointegration because IN preferentially integrate into the host chromosome with no uracils present because uracil causes DNA distortions less favored by IN [321].
Uracilation is especially important in myeloid cells since they have a low dNTP pool. This favors ribonucleotide incorporation by RT, as well as dUTP, a metabolic intermediate during pyrimidine synthesis [254, 255]. This uracil can either protect from autointegration or it can be mutagenic. Usually single base pair misincorporation is repaired by the cellular base excision repair machinery (BER) but macrophages seem to have a lower repair rate because of a reduced RNAse H2 activity [255]. This makes sense since the DNA repair machinery is more important in cycling cells compared to non-dividing cells. The misincorporation of ribonucleotides also slows down the kinetics of reverse transcription. It is worth mentioning that in the absence of SAMHD1 mediated by Vpx, significantly reduces the sensitivity of HIV-1 to nucleoside reverse transcription inhibitors (NRTIs) [322].
Considering these data it makes sense to assume that not only TREX1 is used by HIV-1 as a facilitator to establish an infection without detection but also RNase H2 could play a similar role. As mentioned above RNASEH2B knockout mice are not viable because of an accumulation or incorporate ribonucleotides in the chromosome leading to genomic instability and DNA damage [239]. It is not known if the incorporation of ribonucleotides into the HIV-1 cDNA would cause increased mutations and if the mutations would have an impact on viral replication. But it is reasonable to assume that this could be the case and part of the observed inhibition in the RNASHE2A knockdown experiments in MDDC (chapter 6). This might be another way to hide from detection in dendritic cells by limiting the replication and production of new particles. Another explanation of the observed RNASEH2A phenotype could be that similar to TREX1, RNase H2 complex is able to degrade some aberrant reverse transcription intermediates containing RNA:DNA duplexes.
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Such a hybrid molecule could serve as a PAMP in the cytoplasm to be sensed by an unknown PRR. Knockdown of RNASHE2A would then interfere with the removal of the potential PAMP and type I IFN could be induced leading to the inhibition of HIV-1 like it is the case for TREX1.
The presence of SAMHD1 in myeloid and dendritic cells inhibits full reverse transcription. This prevents HIV-1 from productively infect these cells on one hand and on the other hand it prevents the production of reverse transcription products with the potential of activating the innate immune system as seen above.
Interestingly, when a recombination of SIVRCM and SIVMUS gave rise to SIVCPZ, Vpx from SIVRCM was lost in favor to create a Vif protein, which was able to antagonize the chimp A3G [323]. The SIVCPZ Vif was also able to counteract human A3G, which might have been one of the reasons the virus could cross the species barrier giving rise to HIV-1. Both Vif proteins of SIVRCM and SIVMUS are not able to degrade chimp or human A3G creating the selection pressure for SIVCPZ to use Vpx from SIVRCM to create a new Vif. The Vif of SIVSM - the ancestor of HIV-2 - is already adapted to counteract human A3G and there was no selection pressure to chose between Vpx and Vif and so HIV-2 retained Vpx. SIVSM and HIV-2 are much less pathogenic than HIV-1. One explanation could be that since HIV-2 and SIVSM are able to infect dendritic cells the host can control the virus much better. One report looking at the efficiency of SAMHD1 degradation of HIV-2 Vpx found no correlation with the ability of the infected individuals to control viral replication [324]. When considering that sooty mangabey have high viral load but do not develop AIDS, it seems more likely that it is the host, which tolerates the virus without inducing a strong immune response leading to immune pathologies [18]. One line of evidence is provided by the fact that SAMHD1 restrict HIV-1 reverse transcription in resting T cells [251, 252].
Lymph nodes of HIV-1 infected individuals show a peculiar effect, although only 5%
of CD4+ T cells are actually productively infected, many more T cells go through apoptosis in a by-stander effect. One explanation is that these cells are going through an abortive infection creating reverse transcription intermediates, which are detected by the cells and lead to a caspase 1 depended apoptosis. This process is different from the normal caspase 8-induced apoptosis in that the dying T cell releases inflammatory cytokines causing additional damage on surrounding cells.
This process is called pyrotosis [114]. SAMHD1 could be the reason for the abortive
147
infection in the mostly resting T cells in the lymph node (W.C. Greene, CSHL retroviruses meeting 2013).
Type I IFN is a double-edged sword. On one side it might be one of the causes for HIV-1 induced immune pathologies but on the other hand it is needed to elicit a robust immune response. As mentioned above the last vaccine tested showed first promising results by reducing the risk of infecting by 26% percent. The vaccine was only able to prevent infection but had no effect on viral load or CD4+ T cell count in vaccinated patients and it did not protect from superinfection. For this vaccine a recombinant gp120 was used to induce an immune response [319]. This means even after 30 years of research we still do not know how an effective vaccine has to look like. Clearly the strategy cannot only be to prevent infection at the mucosa but also has to decrease and control viral replication in the patients after infection has taken place. This means we need to find out which epitopes are important to induce a CTL response.
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The data presented in this thesis helps to better understand the interaction between HIV-1 and the innate immune system. A possible next step would be to combine the findings presented here: We would generate dendritic cells knockdown for TREX1 and possibly RNASH2A and challenge these cells with and HIV-1 virus with a capsid variant restricted by TRIM5α and Vpx in trans or cis (chapter 4). HIV-1 could infect these cells and complete reverse transcription thanks to Vpx. The infected cell would present antigen on MHC I and at the same time TRIM5α capsid binding and recognition and the absence of TREX1 and RNASHE2A would activate the cells, allowing the cross talk to CD8+ T cells. That experimental system might allow us to find epitopes which elicit a robust CD8+ T cell response (Figure 7.1). That knowledge could eventually be transformed into a vaccine approach inducing a robust CTL response enabling the immune system to detect and destroy infected cells.
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