HIV Infection Is Associated with Loss of Anti-Inflammatory Alveolar Macrophages.

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163+ anti-inflammatory AM was decreased, whereas CD163-CCR7+ proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers.

Upregulation of Chitinase 1 in Alveolar Macrophages of HIV-Infected Smokers.

Recent studies suggest that HIV infection is an independent risk factor for the development of chronic obstructive pulmonary disease (COPD). We hypothesized that HIV infection and cigarette smoking synergize to alter the function of alveolar macrophages (AMs). To test this hypothesis, global transcriptome analysis was performed on purified AMs from 20 individuals split evenly between HIV-uninfected nonsmokers and smokers and untreated HIV-infected nonsmokers and smokers. Differential expression analysis identified 143 genes significantly altered by the combination of HIV infection and smoking. Of the differentially expressed genes, chitinase 1 (CHIT1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), both previously associated with COPD, were among the most upregulated genes (5- and 26-fold, respectively) in the untreated HIV-infected smoker cohort compared with HIV-uninfected nonsmokers. Expression of CHIT1 and CYP1B1 correlated with the expression of genes involved in extracellular matrix organization, oxidative stress, immune response, and cell death. Using time-of-flight mass cytometry to characterize AMs, a significantly decreased expression of CD163, an M2 marker, was seen in HIV-infected subjects, and CD163 inversely correlated with CYP1B1 expression in AMs. CHIT1 protein levels were significantly upregulated in bronchoalveolar lavage fluid from HIV-infected smokers, and increased CHIT1 levels negatively correlated with lung function measurements. Overall, these findings raise the possibility that elevated CHIT1 and CYP1B1 are early indicators of COPD development in HIV-infected smokers that may serve as biomarkers for determining this risk.

Degradation of SAMHD1 by Vpx Is Independent of Uncoating.

UNLABELLED: Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm. IMPORTANCE: SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

A DNA sequence recognition loop on APOBEC3A controls substrate specificity.

APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus.

Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

The cargo-binding domain of transportin 3 is required for lentivirus nuclear import.

Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-ß family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA (shRNA), we showed that the Drosophila TNPO3 homologue can substitute for its human counterpart and that it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.

ICOS-induced B7h shedding on B cells is inhibited by TLR7/8 and TLR9.

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-gamma and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-gamma production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.

Identification of gene function by cyclical packaging rescue of retroviral cDNA libraries.

Genes regulating responses in mammalian cells are often difficult to identify by functional cloning strategies limited to a single round of selection. Here we describe a strategy, cyclical packaging rescue (CPR), which allows rapid recovery and retransmission of retroviral cDNA libraries. CPR can be used not only with immortalized cell lines such as fibroblasts and Jurkat T cells, but also with primary B lymphocytes, which can be maintained only in short-term cultures. CPR allows for multiple rounds of selection and enrichment to identify cDNAs regulating responses in mammalian cells. Using CPR, five cDNAs were functionally cloned, which conferred protection against tumor necrosis factor alpha (TNFalpha)-induced apoptosis in RelA(-/-) fibroblasts. Three of the genes, RelA, cellular FLICE-like inhibitory protein (c-FLIP), and a dominant-negative mutant of TNF receptor 1 arising through CPR afforded strong protection against apoptosis. Two of the genes identified, Dbs and Fas-associated death domain protein (FADD), previously identified as a proapoptotic molecule, afforded partial protection against TNFalpha-induced apoptosis. These results suggest that CPR is a versatile method that permits functional identification of both wild-type and dominant-negative gene products that regulate cellular responses.