**4.2. APOBEC3**

During HIV-1 infection, incoming viral RNA triggers a TLR7/8-mediated innate immune response, resulting in the production of type I interferon (IFN). In particular IFNα has been shown to be up-regulated after TLR sensing during acute infection with HIV-1 or SIV [23-25]. Accordingly, initial observations *in vitro* revealed that pre-treatment of macrophages with type-I IFN inhibited the replication of HIV-1, indicating that potent inhibitory factors were

The identification of cellular restriction factors and the viral proteins that antagonize those restrictions have stimulated an active area of research that explores crucial mechanisms underlying HIV interference with cellular restriction factors and innate immunity. In this subchapter specific cellular factors with inhibitory activity on HIV replication are discussed

The search for the mechanisms underlying the innate cellular resistance to retroviral infections shown by different non-human primate species, has led to the identification of a cytoplasmic factor that prevented infection of Old World monkeys by HIV-1 [28]. This factor – TRIM5α – was identified as a member of the tripartite motif (TRIM) family of proteins, a large family of cellular proteins with distinct biological activities including innate immune signaling [29]. After its initial identification in rhesus macaques (rhTRIM5α) [28] and owl monkeys (TRIM‐ Cyp) [30], TRIM5α was also identified as a retroviral restriction factor in humans [31, 32] that

Different models have been proposed for retroviral inhibition mediated by TRIM5 proteins [34]. They suggest that these proteins mediate restriction by directly binding to specific determinants in the viral CA protein, blocking HIV replication soon after viral release in host cell cytoplasm. The TRIM proteins family is defined by three domains (RING, B-Box2, and Coiled- Coil), which are present in all members of this family. The N-terminal RING domain possesses E3 ubiquitin ligase activity that is crucial for retrovirus restriction [35, 36]. The B-Box2 and Coiled Coil (CC) domains are thought to contribute to the higher and low order multimerization of TRIM5α, respectively. The TRIM5α also possesses a C-terminal capsid binding domain that mediates specific recognition and restriction of certain retroviruses [37]. The recognition of viral capsid determinants (CA protein) relies on three variable regions present in the C-terminal domain of TRIM5α, and apparently they are equally involved in

Several studies have addressed the mechanisms by which TRIM5α protein prevents viral infection and different models have been proposed to explain this restriction. The "accelerated uncoating" model was based on the observation that cytosolic CA protein was specifically dissociated in rhTRIM5α-expressing cells [42] leading to the proposal of a "proteasome independent capsid degradation" mechanism. This model suggests that the stripping of capsid protein prevents viral RTC to proceed to subsequent steps in infectious replication cycle, namely the reverse transcription and nuclear import [42]. An alternative model was primarily based on the observation that proteasome inhibitors allows reverse transcription and integra‐ tion, without affecting the TRIM5α-mediated restriction [43, 44]. Accordingly, a "two-step

induced after IFN exposure [26, 27]. Most of them are still uncharacterized.

including how viral-encoded proteins counteract these factors.

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

is induced by both type I and type II IFN [33].

retrovirus recognition and restriction [38-41].

**4.1. TRIM5α**

One important form of intrinsic immunity against retroviral infections is provided by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family proteins, and particularly by human APOBEC3G (A3G) and APOBEC3F (A3F) [46-49]. These two proteins are cellular antiretroviral factors that possess inhibitory activity against HIV-1 replication [22, 48, 50].

APOBEC proteins act on single-stranded DNA or RNA substrates and their main function is to induce alterations in the nucleotide sequence through cytidine deamination, converting cytidines to uridines (C to U) or deoxycytidines (dC) to deoxyuridines (dU).

The A3G protein, which expression seems to be regulated at a transcriptional level through NFAT and IRF binding to specific sites located in A3G promoter region [51, 52], is packed inside newly formed HIV-1 virions by a specific interaction with the amino-terminal region of NC domain of HIV-1 Gag polyprotein [53-57]. As expected due to the interaction with NC, A3G is present in viral core as a ribonucleoprotein complex together with genomic RNA, NC, IN and Vpr [58]. Interestingly, binding of A3G to HIV genomic RNA led to inactivation of deaminase activity, while the action of HIV RNase H, which degrades the RNA chain during reverse transcription activates its enzymatic activity [58]. After viral entry into a new cell and during reverse transcription, the released A3G targets the minus-strand DNA product and induces a dC to dU deamination resulting in a dG to dA hypermutation in the HIV-1 doublestranded DNA genome of the replicating virus. This hypermutation activity ultimately introduces mutations and stop codons that disrupt the normal expression and function of viral proteins [46, 59]. A3G can also interfere directly with viral reverse transcriptase preventing RT-dependent cDNA elongation independently of deaminase activity [60]. Finally there is also evidence suggesting that A3G reduces the integration of HIV-1 DNA by interfering with PIC functions [61, 62]. In addition to A3G, also A3F seems to exhibit inhibitory activity against HIV-1 replication [47, 49, 63, 64].

Despite their ability to hinder HIV replication, these proteins only show their potent inhibitory effect with HIV-1 mutants lacking a functional *vif* gene, since the Vif protein expressed by wildtype HIV-1 blocks the function of these host cell proteins [50, 65-70]. Basically, Vif binds to A3G in the cytoplasm of infected cell and directs it for polyubiquitination and proteasomal degradation, preventing its inclusion into the newly formed virions thus overcoming the inhibition of viral replication mediated by A3G [67-69, 71]. A cellular E3 ubiquitin ligase complex consisting of cullin5, elonginB, elonginC and RING finger proteins that binds an E2 ubiquitin-conjugated enzyme, induces the polyubiquitination of A3G. This complex is recruited by Vif that connects it to its substrate inducing the polyubiquitination of A3G [67-69, 71-73]. Additionally, Vif also interferes with the translation of A3G mRNA, reducing its intracellular pool [72, 74].

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

#### **4.3. Tetherin/ BST-2**

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

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 impairs the transport of newly synthetized tetherin by sequestering it within the trans-Golgi network [104-106]. Additionally, Vpu might block the recycling of tetherin after its internaliza‐ tion from the lipid rafts [104, 106, 107]. Finally, it was also proposed that Vpu might directly internalize tetherin from cell membrane [108-110]. Interestingly, it was observed that treat‐ ment with proteasomal inhibitors lead to increased levels of tetherin and loss of Vpu-mediat‐ ed enhancement of HIV-1 release. These results suggest that the Vpu-induced downregulation of tetherin might at least in part involve proteasomal degradation of the restriction factor [111-113]. The exact mechanisms of tetherin down-modulation from cell surface, intracellular sequestration or degradation remain to be determined. These three distinct mechanisms may act cooperatively counteracting tetherin to varying degrees in different cellularcontexts.Regardless themodelthatispreferentiallyobserved,bindingofVputotetherin through TM-TM interaction seems to be crucial for Vpu antagonism of the restriction factor [108, 111, 114, 115].

Despite the wide cellular distribution of tetherin and the need to counteract its viral restriction action, most primate lentiviruses do not contain a *vpu* gene. Some (e.g. SIVsmm, SIVmac, and SIVagm) use their Nef proteins to antagonize tetherin function [116-118]. This is not surprising since Nef protein - a myristoylated protein coded by *nef* gene essential for HIV replication *in vivo* - is known to act as an adaptor protein interacting with different cellular proteins. Through these interactions Nef manipulates cellular trafficking, signal transduction and gene expres‐ sion in HIV infected cells (reviewed in [119]). Apparently, Nef targets the cytoplasmic tail of tetherin reducing its expression at host cell membrane [116, 118]. In alternative to Nef, HIV-2 relies on its envelope glycoprotein Env to antagonize tetherin. The proposed mechanism suggests that Env interacts directly with the ectodomain of tetherin, sequestering it away from sites of virus budding and targeting it to clathrin-mediated endocytosis [120].

Besides the referred lentiviruses, the antiviral activity of tetherin was also demonstrated against a broad range of unrelated viruses, such as filoviruses [87, 88], arenaviruses [88] and herpesviruses [121, 122]. For some of these viruses specific viral encoded antagonists has been described. For example, human herpesviruses 8 (HHV-8, also known as Kaposi's Sarcoma herpesvirus) uses K5/MIR2 - a viral protein belonging to the membrane-associated RING-CH ubiquitin ligase family - to ubiquitinate tetherin and target it for degradation [121]. In Ebola virus - a filovirus associated with hemorrhagic fever outbreaks - the tetherin-mediated restriction is counteracted by viral envelope glycoprotein [123] in a process similar to the described sequestration of tetherin by HIV-2 Env.
