**4.5. MxB**

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

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

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

immunity.

cells during antigen presentation process within lymph nodes (reviewed in [129]).

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

The myxovirus resistance (Mx) genes were discovered in the 1960s when it was observed that wild mice were resistant to influenza viruses, whereas inbred mice were susceptible [152]. This trait was later mapped to a locus on mouse chromosome 16 [153-156]. Mx family proteins are found in almost all vertebrates, demonstrating their evolutionary importance for host organ‐ isms [157]. Humans Mx gene resides on chromossome 21 [158] and encodes two proteins, called MxA and MxB, that belong to the family of dynamin-like large GTPases. The MxA protein has been recognized as a potent cell restriction factor with antiviral activity against pathogenic DNA and RNA viruses [159].

The X-ray crystal structure of human MxA showed that this protein can be divided into a globular GTPase head, a largely C-terminal α-helical stalk domain and a series of α-helices found in sequences adjacent to these domains which fold in the protein tertiary structure to form the bundle signaling element (BSE) [160]. On the basis of sequence homology and computer modeling the predicted structure of MxB is almost superimposable with that of MxA, having 63% amino acid sequence identity.

In contrast to human MxA protein that inhibits a variety of viruses [161], MxB was initially described as lacking antiviral activity against influenza or vesicular stomatitis virus [162]. Instead, MxB was solely related to cellular functions, such as regulating nuclear import and cell-cycle progression [163, 164].

This view was challenged in 2011, when Schoggings and collaborators addressed an overex‐ pression screening to test the antiviral activity of more than 380 human interferon stimulated gene (ISGs) products against a panel of viruses, where they first uncovered an antiviral activity of human MxB against HIV-1 [165].

More recently, three additional studies [166-168] showed that MxB overexpression potently reduces the permissiveness of the cells in a single-cycle HV-1 infection assay. They also demonstrate that silencing MxB expression reduced the inhibitory potency of the interferonα demonstrating its importance in the interferon-mediated response against the early steps of HIV-1 infection.

The next step was to understand which specific post-entry event of the HIV replication cycle was affected by MxB expression. Recent studies agreed that MxB expression potently inhibited HIV-1 infection after reverse transcription but before integration [166-168]. So MxB might be interfering with one or more of the following processes: 1) HIV-1 uncoating; 2) nuclear import of the HIV-1 PIC; or 3) nuclear maturation of the PIC.

Fricke and colleagues [169] suggested a model in which MxB binds to the HIV-1 core in the cytoplasm of the cell and prevents the uncoating process of HIV-1 through stabilization of incoming viral capsides. In addition, they demonstrated that MxB requires capsid binding and oligomerization for effective restriction.

More recently, Matreyek et al. [170] observed that MxB restricts HIV-1 after DNA synthesis at steps that are coincident with PIC nuclear import and integration.

HIV-1 RNA is reverse transcribed into double stranded linear DNA and carries a fraction of the viron CA protein [171, 172]. HIV-1 CA protein is known to play a central role in mediating physical interactions with several host proteins involved in the post-entry step of infection. Some identified residues of CA involved in binding to cyclophilin A (CypA), TRIM5α, TNP03, CPSF6, NUP153 and NUP358/RanBP2 are also critical for the sensitivity of HIV-1 to the antiviral action of MxB. Results obtained by Liu and colleagues indicate that both silencing of CypA expression or disruption of the CA-CypA interaction by addition of cyclosporine A abrogated the antiviral activity of MxB, thus CypA binding to the HIV-1 CA appears to be required for MXB restriction. Furthermore, results obtained by diverse groups indicate that CA mutations counteracted MxB restriction [165-168, 170].

The viral integrase (IN) protein processes the long terminal repeat (LTR) ends of the viral DNA to yield the integration-competent PIC, which subsequently transports the viral DNA into the nucleus for IN-mediated integration [173]. Matreyek and collaborators [170] found evidence for an additional block in the formation of 2-LTR circular viral DNA (that are only present in the nucleus, and thus have been utilized as a marker of nuclear entry of viral DNA [174]). In contrast, results obtained by Liu and collaborators [167] showed that MxB reduces the levels of integrated HIV-1 DNA, though it does not affect the amount of 2-LTR circles. They con‐ cluded that MxB impairs the integration step and spares the nuclear entry of viral DNA.

Apparently, MxB antiviral activity is independent of its GTPase active site residues or stalk domain Loop4 (both previously shown to be necessary for MxA function) that confer func‐ tional oligomerization to related dynamin family proteins [166, 168]. There are two locations in MxB that exhibit the greatest sequence dissimilarity with MxA. The first one is Loop4 that is not critical for MxB antiviral activity but is important for the MxA inhibition of Influenza A and Thogotovirus infection [170, 175]. The other part of MxB with greatest dissimilarity to MxA is the N-terminal region. The specific particular functions conferred by this region are particularly important for MxB activity and consequent HIV-1 restriction [170].

In a global perspective, the post-entry step of HIV-1 replication cycle appears to be quite vulnerable to the actions of IFN-inducible restriction factors: TRIM5α, APOBEC3 proteins, SAMHD1 and, more recently, MxB use distinct mechanisms to prevent integration of this pathogenic virus in host genome. Certainly it will continue to be of interest to the scientific community the study of restriction factors of viral infection by antiviral host factors due to its impact in many areas. These findings raises hope as a potential clinical and epidemiological relevant approach which could be exploited to control HIV infections and AIDS.
