**4.4. SAMHD1**

Besides A3G and A3F proteins, the human genome also contains genes encoding five others members of the APOBEC3 family. However, of these five additional genes, apparently only three (APOBEC3A, APOBEC3B and APOBEC3C) are expressed in human cells. Recent data shows that APOBEC3A is recruited at post-entry HIV-1 replication complexes [75-79]. Its expression is induced in monocyte-derived macrophages (MDM) by interferon-alpha (IFN-α) and it seems to promote resistance to HIV-1 infection in MDM [75]. The APOBEC3C protein is a weak inhibitor of wild-type or *vif*-deficient HIV-1 [63, 64, 80] although it was described, together with APOBEC3B, as a potent inhibitor of simian immunodeficiency virus (SIV) replication [81]. As for A3G, the APOBEC3B protein is also packed inside HIV-1 virions due to a specific interaction with the NC protein. It induces a potent inhibition of HIV-1 replication and it seems to be resistant to HIV-1 Vif protein [82]. However, APOBEC3B is expressed at

In early 2008, an additional restriction factor dubbed Tetherin, previously referred to as BST-2, CD317 or HM1.24, was described [83, 84]. The main function of this IFN-induced protein [85, 86] remained elusive until it was identified as an intrinsic antiviral factor that restricts the egress of HIV and other enveloped viruses by tethering mature virions to the host cell membrane [83, 84, 87-91]. Tetherin is a type II membrane protein highly expressed at the plasma membrane of B cells at all differentiation stages, bone-marrow CD34+ cells and T-cells [92]. It has an unusual topology consisting of an amino-terminal cytoplasmic tail (CT), followed by a transmembrane region that anchors tetherin to the plasma membrane and a coiled-coil extracellular domain that is also linked to the plasma membrane by a carboxy-terminal glycophosphatidylinositol (GPI) anchor [93, 94]. Due to the presence of this GPI anchor, tetherin is mainly located in cholesterol-rich microdomains also referred as "lipid rafts". Tetherin is involved (through the CT domain) in the organization of subapical actin cytoske‐ leton in polarized epithelial cells [95] and unlike other GPI-anchored proteins, is endocytosed

Coincident with the identification of tetherin as an antiviral factor, it was also found that it was the target of the HIV-1 accessory protein Vpu, providing a plausible mechanism for the wellestablished but ill-defined, virus-release function of Vpu [83]. The Vpu is a small transmem‐ brane (TM) protein encoded by the *vpu* gene present in the genomes of HIV-1 and some SIV strains, but absent in HIV-2. It is anchored to the plasma membrane of the infected cell by its amino-terminal region. Initial studies showed that Vpu protein besides its ability to degrade CD4 protein [97], was also required for efficientreplication of HIV-1 in some cell types and that the restriction factor counteracted by Vpu was a protein located at cell surface [16, 98-101]. This factor was found to be IFNα-inducible and showed the ability to block the release of Vpudefective virions by directly tethering them to the plasma membrane of virus-producer cells. The trapped virions are subsequently internalized by endocytosis and probably degraded in lysosomes [83, 85]. Remarkably, the lipid rafts localization of tetherin is coincident with the preferential site for budding and egress of enveloped viruses [102, 103], providing further explanation for the mechanism by which tetherin blocks virion release. Several aspects of the Vpu-mediated antagonism of tetherin are still controversial. It was initially proposed that Vpu

very low levels in human tissues, in contrast to A3G and A3F [82].

82 Trends in Basic and Therapeutic Options in HIV Infection - Towards a Functional Cure

from lipid rafts in a clathrin-mediated pathway [96].

**4.3. Tetherin/ BST-2**

Myeloid-lineage cells, including monocytes, dendritic cells (DCs) and macrophages, play a multifaceted role in HIV-1 initial infection and viral dissemination during acute infection. In particular, the interactions between HIV and DCs are connected with all aspects of HIV infection *in vivo*, including transmission, pathogenesis and immune control (recently reviewed in [124]). DCs exposed to HIV during sexual transmission help viral dissemination and systemic infection by two distinct mechanisms: by becoming productively infected or by transferring HIV to CD4+ T cells during immunologic synapse (IS), even in the absence of DCs infection [125-127].

AlthoughDCs canbeinfected,HIVreplicationisgenerallylessproductivecomparedwithCD4+ T cells. Nevertheless, extensive viral replication takes place once DCs come into contact with CD4+ T cells in lymphoid tissue in the context of IS [128]. This implies that HIV must be able to evade DC's innate immune sensing and endolysosomal degradation and then make use of DC maturation and migration to draining lymph nodes to be transmitted to highly susceptible T cells during antigen presentation process within lymph nodes (reviewed in [129]).

Infection by DNA or RNA viruses triggers innate immune responses when host recognizes specific viral molecular structures (e.g. nucleic acid and surface glycoprotein), called patternassociated molecular patterns (PAMPs) [130-132]. These PAMPs are recognized by patternrecognition receptors (PRRs), such as Toll-like receptors (TLRs), RIG-I-like helicases (RLH), and cytosolic DNA sensor proteins. Inside the cytoplasm, viral nucleic acid can be detected by different PRRs depending on the cell type. For example, TLR7 and TLR9 are responsible for detection of viral RNA and DNA, respectively, in plasmacytoid dendritic cells (pDCs), whereas RLHs detect viral RNA in conventional DCs, macrophages and fibroblasts [133, 134]. The recognition of PAMPs by the PRRs activates several transcription factors, namely nuclear factor-κappa binding (NF-κB) and IFN regulatory factors (IRFs). This activation leads to the production of pro-inflammatory cytokines and type-I IFNs (IFN-α and IFN-β), respectively (reviewed in [130]). The production of type-I IFNs induces the expression of hundreds of interferon-stimulated genes (ISGs) [135], providing crucial mechanisms of antiviral defense by inhibiting viral replication and spread. For example, during HIV Infection, viral singlestranded RNA (ssRNA) is recognized by TLR7/8 initiating anti-HIV immune response by inducing type I IFN. However, as a well-adapted human pathogen HIV must be able to avoid – at least in part – these cell sensing mechanisms in order to evade host innate immunity.

Sterile alpha motif (SAM) and histidine/aspartic acid (HD) domain-containing protein 1 (SAMHD1), an analogue of the murine IFN-y-induced gene Mg11 [136], was identified as a HIV-1 restriction factor that blocks early-stage virus replication in DCs and other myeloid cells [137, 138]. It acts by depleting the intracellular pool of deoxynucleoside triphosphates (dNTP), thus impairing HIV-1 reverse transcription and productive infection [139-141]. The expected lower replication in DCs may enable HIV-1 to avoid intracellular viral sensor that would otherwise trigger IFN-mediated antiviral immunity [142, 143]. It seems that while SAMHD1 effectively renders DCs less permissive to HIV-1 infection, it is somewhat paradoxically responsible for the HIV-1 evasion of immune sensing and subsequent poor priming of adaptive immunity.

HIV-2 brings in a new and interesting element: Vpx (an accessory protein encoded by *vpx* gene, present in SIVsm/SIVmac and HIV-2), that is believed to have originated by duplication of the common *vpr* gene present in primate lentiviruses [144], possibly to compensate for a theor‐ ised low HIV-2 RT affinity for dNTPs [145, 146]. This accessory protein antagonizes the effect of SAMHD1 by targeting it for proteasomal degradation using the host cell E3 ubiquitin ligase complex, in which Vpx interacts with the DCAF1 subunit of the CUL4A/DDB1 ubiquitin ligase to degrade SAMHD1 via the proteasome [137, 139, 147, 148]. The degradation of SAMHD1 renders HIV-2-infected DCs much more permissive to productive infection and viral replica‐ tion,allowingfasteraccumulationoffulllengthviralDNA[148].This results inawidelypositive DC-specific effect in the innate immune sensing of HIV-2 infection [143, 146, 148, 149] and it may be related to the lower viral load and slower progression to AIDS that is characteristic of HIV-2 infection (reviewed in [150]). The immunologically positive effects of Vpx was also demonstrated in monocyte-derived DCs (MMDCs) infected with HIV-1 where an increased type I IFN production and up-regulation of CD86 was only observed in the presence of Vpx [146]. Hence, by avoiding productive infection of MDDCs through preservation of SAMHD1 function, HIV-1 may also control viral antigen presentation, resulting in qualitatively or quantitatively minor CD8+ and CD4+ responses [146, 149, 151]. Furthermore, individuals with low SAMHD1 activity or silenced SAMHD1, present an enhanced immune response to HIV-1 infection, as previously hypothesised [139, 143] [139, 143] and demonstrated [146].
