Transplantation and immigration: Comparing infectious complications and outcomes between foreign-born and US-born kidney transplant recipients in Minnesota.

BACKGROUND: Foreign-born kidney transplant recipients (FBKTRs) are at increased risk for reactivation of latent infections that may impact outcomes. We aimed to compare the etiology of infections and outcomes between FBKTR and United States KTRs (USKTR). METHODS: We performed a retrospective study of patients who underwent kidney transplantation between January 1, 2014 and December 31, 2018 at two transplant centers in Minnesota. Frequency and etiology of infections as well as outcomes (graft function, rejection, and patient survival) at 1-year post-transplant between FBKTR and USKTR were compared. RESULTS: Of the 573 transplant recipients, 124 (21.6%) were foreign-born and 449 (78.4%) US-born. At least one infection occurred in 411 (71.7%) patients (38.2% bacterial, 55% viral, 9.4% fungal). Viral infections were more frequent in FBKTR, particularly BK viremia (38.7% vs. 21.2%, p < .001). No statistical differences were found for bacterial or fungal infections; no parasitic infections were identified in either group. No geographically-restricted infections were noted aside from a single case of Madura foot in a FBKTR. Rejection episodes were more common in USKTR (p = .037), but stable/improving graft function (p = .976) and mortality (p = .451) at 1-year posttransplantation were similar in both groups. After adjusting for covariates, previous transplantation was associated with a higher number of infections (IRR 1.35, 95% confidence intervals 1.05-1.73, p = .020). CONCLUSION: Although viral infections were more frequent in FBKTR, overall frequency and etiology of most infections and outcomes were similar between FBKTR and USKTR suggesting that comprehensive transplant care is providing timely prevention, diagnosis, and treatment of latent infections in FBKTR.

Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis.

Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer.

Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G.

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the α1-ß1, ß2-α2 and ß4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.

The PKC/NF-κB signaling pathway induces APOBEC3B expression in multiple human cancers.

Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in many cancers. Human papillomavirus infection accounts for APOBEC3B upregulation in cervical and head/neck cancers, but the mechanisms underlying nonviral malignancies are unclear. In this study, we investigated the signal transduction pathways responsible for APOBEC3B upregulation. Activation of protein kinase C (PKC) by the diacylglycerol mimic phorbol-myristic acid resulted in specific and dose-responsive increases in APOBEC3B expression and activity, which could then be strongly suppressed by PKC or NF-κB inhibition. PKC activation caused the recruitment of RELB, but not RELA, to the APOBEC3B promoter, implicating noncanonical NF-κB signaling. Notably, PKC was required for APOBEC3B upregulation in cancer cell lines derived from multiple tumor types. By revealing how APOBEC3B is upregulated in many cancers, our findings suggest that PKC and NF-κB inhibitors may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes, such as drug resistance and metastasis.

The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches.

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-ß and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamic simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a wobble model to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, how pathogens may evolve to escape innate host defenses.

APOBEC3 multimerization correlates with HIV-1 packaging and restriction activity in living cells.

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including human immunodeficiency virus type 1 (HIV-1). Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.