In contrast to activated CD4+ T cells, resting human CD4+ T cells circulating in blood are highly resistant to infection with human immunodeficiency virus (HIV). Whether the inability of HIV to infect these resting CD4+ T cells is due to the lack of a key factor, or alternatively reflects the presence of an efficient mechanism for defence against HIV, is not clear. Here we show that the anti-retroviral deoxycytidine deaminase APOBEC3G strongly protects unstimulated peripheral blood CD4+ T cells against HIV-1 infection. In activated CD4+ T cells, cytoplasmic APOBEC3G resides in an enzymatically inactive, high-molecular-mass (HMM) ribonucleoprotein complex that converts to an enzymatically active low-molecular-mass (LMM) form after treatment with RNase. In contrast, LMM APOBEC3G predominates in unstimulated CD4+ T cells, where HIV-1 replication is blocked and reverse transcription is impaired. Mitogen activation induces the recruitment of LMM APOBEC3G into the HMM complex, and this correlates with a sharp increase in permissivity for HIV infection in these stimulated cells. Notably, when APOBEC3G-specific small interfering RNAs are introduced into unstimulated CD4+ T cells, the early replication block encountered by HIV-1 is greatly relieved. Thus, LMM APOBEC3G functions as a potent post-entry restriction factor for HIV-1 in unstimulated CD4+ T cells. Surprisingly, sequencing of the reverse transcripts slowly formed in unstimulated CD4+ T cells reveals only low levels of dG dA hypermutation, raising the possibility that the APOBEC3G-restricting activity may not be strictly dependent on deoxycytidine deamination

RNA granules ͉ Ro ribonucleoproteins ͉ prespliceosomes T he intrinsic antiretroviral factor APOBEC3G (A3G) is highly active against HIV-1 and other retroviruses (1). Incorporation of A3G into budding HIV-1 virions promotes extensive mutation of nascent HIV-1 DNA formed by reverse transcription in the next round of infection (2-5). However, HIV-1 counters these effects of A3G with virion infectivity factor (Vif), which accelerates proteasome-mediated degradation of A3G (6-11) and partially impairs de novo synthesis of A3G (6, 12). These two actions in virus-producing cells effectively deplete intracellular A3G, making the enzyme unavailable for virion encapsidation. Resting CD4 T cells and monocytes, which are refractory to HIV-1 infection, express only the low-molecular-mass (LMM) form of A3G (13). siRNAmediated knockdown of LMM A3G expression in resting CD4 T cells renders these cells permissive for HIV-1 infection, indicating that LMM A3G functions as a potent postentry restriction factor for HIV-1 (13). Conversely, resting CD4 T cells in lymphoid tissues are permissive for HIV-1 infection, and A3G is predominantly in high-molecular-mass (HMM) complexes in these cells (14) because of the lymphoid microenvironment. Locally produced cytokines, including IL-2 and IL-15, and cell-cell interactions in lymphoid tissues stimulate assembly of the HMM A3G complexes (14) and confer permissiveness for HIV-1 infection.The genes encoding A3G and other APOBEC3 (A3) family members are clustered on human chromosome 22 (15). During mammalian evolution, this locus expanded from a single gene in mice to eight genes (A3A-H) in primates (15, 16). These genes apparently have been modulated by repeated episodes of strong Abbreviations: HMM, high-molecular-mass; LMM, low-molecular-mass; RNP, ribonucleoprotein; A3G, APOBEC3G; L1, long interspersed nucleotide elements 1; TAP, tandem affinity purification; IP, immunoprecipitation; co-IP, coimmunoprecipitation; scAlu, small cytoplasmic Alu; PB, processing body. ¶ To whom correspondence should be addressed. E-mail: wgreene@gladstone.ucsf.

We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinfl ammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3infl ammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Effi cacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the signifi cant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infi ltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specifi c T-cell transfer. SIGNIFICANCE :Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-infl ammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the effi cacy of adoptive T-cell transfer.

APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older “mature” A3G already residing in high-molecular-mass RNA–protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.

Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.