Nathan C. Gaddis

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

Viruses have developed diverse non-immune strategies to counteract host-mediated mechanisms that confer resistance to infection. The Vif (virion infectivity factor) proteins are encoded by primate immunodeficiency viruses, most notably human immunodeficiency virus-1 (HIV-1). These proteins are potent regulators of virus infection and replication and are consequently essential for pathogenic infections in vivo. HIV-1 Vif seems to be required during the late stages of virus production for the suppression of an innate antiviral phenotype that resides in human T lymphocytes. Thus, in the absence of Vif, expression of this phenotype renders progeny virions non-infectious. Here, we describe a unique cellular gene, CEM15, whose transient or stable expression in cells that do not normally express CEM15 recreates this phenotype, but whose antiviral action is overcome by the presence of Vif. Because the Vif:CEM15 regulatory circuit is critical for HIV-1 replication, perturbing the circuit may be a promising target for future HIV/AIDS therapies.

The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif

The human protein apolipoprotein B mRNA-editing enzyme–catalytic polypeptide-like-3G (APOBEC3G), also known as CEM-15, mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates. Because DNA deamination takes place after virus entry into target cells, APOBEC3G function is dependent on its association with the viral nucleoprotein complexes that synthesize cDNA and must therefore be incorporated into virions as they assemble in infected cells. Here we show that the HIV-1 virion infectivity factor (Vif) protein protects the virus from APOBEC3G-mediated inactivation by preventing its incorporation into progeny virions, thus allowing the ensuing infection to proceed without DNA deamination. In addition to helping exclude APOBEC3G from nascent virions, Vif also removes APOBEC3G from virus-producing cells by inducing its ubiquitination and subsequent degradation by the proteasome. Our findings indicate that pharmacologic strategies aimed at stabilizing APOBEC3G in HIV-1 infected cells should be explored as potential HIV/AIDS therapeutics.

Unusual Polymorphisms in Human Immunodeficiency Virus Type 1 Associated with Nonprogressive Infection

ABSTRACT Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5-Δ32, CCR2-64I, andSDF-1-3′A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases.

GATA transcription factor required for immunity to bacterial and fungal pathogens.

In the past decade, Caenorhabditis elegans has been used to dissect several genetic pathways involved in immunity; however, little is known about transcription factors that regulate the expression of immune effectors. C. elegans does not appear to have a functional homolog of the key immune transcription factor NF-κB. Here we show that that the intestinal GATA transcription factor ELT-2 is required for both immunity to Salmonella enterica and expression of a C-type lectin gene, clec-67, which is expressed in the intestinal cells and is a good marker of S. enterica infection. We also found that ELT-2 is required for immunity to Pseudomonas aeruginosa, Enterococcus faecalis, and Cryptococcus neoformans. Lack of immune inhibition by DAF-2, which negatively regulates the FOXO transcription factor DAF-16, rescues the hypersusceptibility to pathogens phenotype of elt-2(RNAi) animals. Our results indicate that ELT-2 is part of a multi-pathogen defense pathway that regulates innate immunity independently of the DAF-2/DAF-16 signaling pathway.

Comprehensive Investigation of the Molecular Defect in vif-Deficient Human Immunodeficiency Virus Type 1 Virions

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1) in primary blood lymphocytes, certain T-cell lines (nonpermissive cells), and most likely in vivo is highly dependent on the virally encoded Vif protein. Evidence suggests that Vif acts late in the viral life cycle during assembly, budding, and/or maturation to counteract the antiviral activity of the CEM15 protein and possibly other antiviral factors. Because HIV-1 virions produced in the absence of Vif are severely restricted at a postentry, preintegration step of infection, it is presumed that such virions differ from wild-type virions in some way. In the present study, we established a protocol for producing large quantities of vif-deficient HIV-1 (HIV-1/Δvif) from an acute infection of nonpermissive T cells and performed a thorough examination of the defect in these virions. Aside from the expected lack of Vif, we observed no apparent abnormalities in the packaging, modification, processing, or function of proteins in Δvif virions. In addition, we found no consistent defect in the ability of Δvif virions to perform intravirion reverse transcription under a variety of assay conditions, suggesting that the reverse transcription complexes in these particles can behave normally under cell-free conditions. Consistent with this finding, neither the placement of the primer tRNA3Lys nor its ability to promote reverse transcription in an in vitro assay was affected by a lack of Vif. Based on the inability of this comprehensive analysis to uncover molecular defects in Δvif virions, we speculate that such defects are likely to be subtle and/or rare.

Further Investigation of Simian Immunodeficiency Virus Vif Function in Human Cells

ABSTRACT Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two vif-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.

Unfolded Protein Response Genes Regulated by CED-1 Are Required for Caenorhabditis elegans Innate Immunity

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.

Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence.

We conducted a 1000 Genomes–imputed genome-wide association study (GWAS) meta-analysis for nicotine dependence, defined by the Fagerström Test for Nicotine Dependence in 17 074 ever smokers from five European-ancestry samples. We followed up novel variants in 7469 ever smokers from five independent European-ancestry samples. We identified genome-wide significant association in the alpha-4 nicotinic receptor subunit (CHRNA4) gene on chromosome 20q13: lowest P=8.0 × 10−9 across all the samples for rs2273500-C (frequency=0.15; odds ratio=1.12 and 95% confidence interval=1.08–1.17 for severe vs mild dependence). rs2273500-C, a splice site acceptor variant resulting in an alternate CHRNA4 transcript predicted to be targeted for nonsense-mediated decay, was associated with decreased CHRNA4 expression in physiologically normal human brains (lowest P=7.3 × 10−4). Importantly, rs2273500-C was associated with increased lung cancer risk (N=28 998, odds ratio=1.06 and 95% confidence interval=1.00–1.12), likely through its effect on smoking, as rs2273500-C was no longer associated with lung cancer after adjustment for smoking. Using criteria for smoking behavior that encompass more than the single ‘cigarettes per day’ item, we identified a common CHRNA4 variant with important regulatory properties that contributes to nicotine dependence and smoking-related consequences.

Assessment of genotype imputation performance using 1000 Genomes in African American studies.

Genotype imputation, used in genome-wide association studies to expand coverage of single nucleotide polymorphisms (SNPs), has performed poorly in African Americans compared to less admixed populations. Overall, imputation has typically relied on HapMap reference haplotype panels from Africans (YRI), European Americans (CEU), and Asians (CHB/JPT). The 1000 Genomes project offers a wider range of reference populations, such as African Americans (ASW), but their imputation performance has had limited evaluation. Using 595 African Americans genotyped on Illumina’s HumanHap550v3 BeadChip, we compared imputation results from four software programs (IMPUTE2, BEAGLE, MaCH, and MaCH-Admix) and three reference panels consisting of different combinations of 1000 Genomes populations (February 2012 release): (1) 3 specifically selected populations (YRI, CEU, and ASW); (2) 8 populations of diverse African (AFR) or European (AFR) descent; and (3) all 14 available populations (ALL). Based on chromosome 22, we calculated three performance metrics: (1) concordance (percentage of masked genotyped SNPs with imputed and true genotype agreement); (2) imputation quality score (IQS; concordance adjusted for chance agreement, which is particularly informative for low minor allele frequency [MAF] SNPs); and (3) average r2hat (estimated correlation between the imputed and true genotypes, for all imputed SNPs). Across the reference panels, IMPUTE2 and MaCH had the highest concordance (91%–93%), but IMPUTE2 had the highest IQS (81%–83%) and average r2hat (0.68 using YRI+ASW+CEU, 0.62 using AFR+EUR, and 0.55 using ALL). Imputation quality for most programs was reduced by the addition of more distantly related reference populations, due entirely to the introduction of low frequency SNPs (MAF≤2%) that are monomorphic in the more closely related panels. While imputation was optimized by using IMPUTE2 with reference to the ALL panel (average r2hat = 0.86 for SNPs with MAF>2%), use of the ALL panel for African American studies requires careful interpretation of the population specificity and imputation quality of low frequency SNPs.

Cis-Expression Quantitative Trait Loci Mapping Reveals Replicable Associations with Heroin Addiction in OPRM1.

BACKGROUND No opioid receptor, mu 1 (OPRM1) gene polymorphisms, including the functional single nucleotide polymorphism (SNP) rs1799971, have been conclusively associated with heroin/other opioid addiction, despite their biological plausibility. We used evidence of polymorphisms altering OPRM1 expression in normal human brain tissue to nominate and then test associations with heroin addiction. METHODS We tested 103 OPRM1 SNPs for association with OPRM1 messenger RNA expression in prefrontal cortex from 224 European Americans and African Americans of the BrainCloud cohort. We then tested the 16 putative cis-expression quantitative trait loci (cis-eQTL) SNPs for association with heroin addiction in the Urban Health Study and two replication cohorts, totaling 16,729 European Americans, African Americans, and Australians of European ancestry. RESULTS Four putative cis-eQTL SNPs were significantly associated with heroin addiction in the Urban Health Study (smallest p = 8.9 × 10(-5)): rs9478495, rs3778150, rs9384169, and rs562859. Rs3778150, located in OPRM1 intron 1, was significantly replicated (p = 6.3 × 10(-5)). Meta-analysis across all case-control cohorts resulted in p = 4.3 × 10(-8): the rs3778150-C allele (frequency = 16%-19%) being associated with increased heroin addiction risk. Importantly, the functional SNP allele rs1799971-A was associated with heroin addiction only in the presence of rs3778150-C (p = 1.48 × 10(-6) for rs1799971-A/rs3778150-C and p = .79 for rs1799971-A/rs3778150-T haplotypes). Lastly, replication was observed for six other intron 1 SNPs that had prior suggestive associations with heroin addiction (smallest p = 2.7 × 10(-8) for rs3823010). CONCLUSIONS Our findings show that common OPRM1 intron 1 SNPs have replicable associations with heroin addiction. The haplotype structure of rs3778150 and nearby SNPs may underlie the inconsistent associations between rs1799971 and heroin addiction.