Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production.

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4(FANCP) deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.

DNA damage repair machinery and HIV escape from innate immune sensing.

Viruses have been long known to perturb cell cycle regulators and key players of the DNA damage response to benefit their life cycles. In the case of the human immunodeficiency virus (HIV), the viral auxiliary protein Vpr activates the structure-specific endonuclease SLX4 complex to promote escape from innate immune sensing and, as a side effect, induces replication stress in cycling cells and subsequent cell cycle arrest at the G2/M transition. This novel pathway subverted by HIV to prevent accumulation of viral reverse transcription by-products adds up to facilitating effects of major cellular exonucleases that degrade pathological DNA species. Within this review we discuss the impact of this finding on our understanding of the interplay between HIV replication and nucleic acid metabolism and its implications for cancer-related chronic inflammation.

Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing.

The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

Comparative proteomic analysis of HIV-1 particles reveals a role for Ezrin and EHD4 in the Nef-dependent increase of virus infectivity.

Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.

Suboptimal provirus expression explains apparent nonrandom cell coinfection with HIV-1.

Despite the ability of primate lentiviruses to prevent infected cells from being reinfected, cell coinfection has occurred in the past and has shaped virus evolution by promoting the biogenesis of heterozygous virions and recombination during reverse transcription. In vitro experiments have shown that cell coinfection with HIV is more frequent than would be expected if coinfection were a random process. A possible explanation for this bias is the heterogeneity of target cells and the preferred infection of a subpopulation. To address this question, we compared the frequency of double-positive cells measured following coincubation with green fluorescent protein (GFP) and DsRed HIV reporter viruses with that of stochastic coinfection calculated as the product of the frequencies of GFP- and DsRed-positive cells upon incubation with either reporter virus. Coinfection was more frequent than would be expected on the grounds of stochastic infection, due to the underestimation of single-infection frequencies, which mathematically decreased the calculated frequency. Indeed, when cells were incubated with either reporter virus, a fraction of the cells were scored as uninfected yet harbored a silent provirus that was reactivated upon coinfection through cross talk between viral elements. When such cross talk was avoided, experimental and calculated coinfection frequencies matched, indicating random coinfection. The proportion of infected cells harboring a silent provirus was estimated from coinfection experiments and was shown to be cell type dependent but independent of the virus entry route.

Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins.

The genomes of all retroviruses encode the Gag Pol and Env structural proteins. Human and simian lentiviruses have acquired non-structural proteins among which Nef plays a major role in the evolution of viral infection towards an immunodeficiency syndrome. Indeed, in the absence of a functional nef gene, primate lentiviruses are far less pathogenic than their wild type counterparts. The multiple protein-protein interactions in which Nef is involved all contribute to explain the role played by Nef in HIV- and SIV-associated disease progression. This review summarizes common and distinct features among Nef proteins and how they contribute to increasing HIV and SIV fitness towards their respective hosts.

Nef-induced CD4 endocytosis in human immunodeficiency virus type 1 host cells: role of p56lck kinase.

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56(lck) kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56(lck) in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56(lck)-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56(lck) with cell surface-expressed CD4. Regardless of the presence of p56(lck), the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.