The HIV-1 Protein Vpr Targets the Endoribonuclease Dicer for Proteasomal Degradation to Boost Macrophage Infection

The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4DCAF1 ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4DCAF1. Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4DCAF1. We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4DCAF1 ubiquitin ligase complex and its subsequent degradation

HIV-1 Vpr Causes Synaptodendritic Damage in Neurons

HIV weakens the immune system by infecting and destroying T-cells, leaving the body vulnerable to infection and the development of AIDS. Conventional treatments for HIV, such as combined anti-rectroviral therapy (cART), fail to prevent the development of HIV-associated neurocognitive disorder (HAND). Neurological dysfunction has been directly related to the invasion of HIV in the central nervous system (CNS). HIV produces neurotoxic proteins, such as the Viral Protein R (Vpr), which contribute to HAND. Astrocytes are the most abundant cells in the brain and an important HIV target. We hypothesize that astrocytes expressing Vpr will cause neuronal damage in our co-culture system. Primary astrocytes were transfected with Vpr plasmid or control (pEGFP or mock) using electroporation. Astrocytes were then co-cultured with cortical neurons. At 48 and 72 hours we collected the primary astrocytes to confirm the Vpr expression via western blot analysis. We then measured structural damage in the neurons using immunofluorescence for cytoskeletal (MAP2, f-actin) and synaptic (synaptophysin) damage. Preliminary results showed strong staining of filamentous actin and MAP2 with weak detection of synaptophysin. The positive control for neurotoxicity (2.8µM acrylamide) showed substantial damage to the cellular structure. Results for Vpr expression are pending. After confirming that the immunofluorescence assays are working with our controls, we expect to detect any synaptodendritic damage in the neurons caused by Vpr in our upcoming experiments

Evolutionary Toggling of Vpx/Vpr Specificity Results in Divergent Recognition of the Restriction Factor SAMHD1

SAMHD1 is a host restriction factor that blocks the ability of lentiviruses such as HIV-1 to undergo reverse transcription in myeloid cells and resting T-cells. This restriction is alleviated by expression of the lentiviral accessory proteins Vpx and Vpr (Vpx/Vpr), which target SAMHD1 for proteasome-mediated degradation. However, the precise determinants within SAMHD1 for recognition by Vpx/Vpr remain unclear. Here we show that evolution of Vpx/Vpr in primate lentiviruses has caused the interface between SAMHD1 and Vpx/Vpr to alter during primate lentiviral evolution. Using multiple HIV-2 and SIV Vpx proteins, we show that Vpx from the HIV-2 and SIVmac lineage, but not Vpx from the SIVmnd2 and SIVrcm lineage, require the C-terminus of SAMHD1 for interaction, ubiquitylation, and degradation. On the other hand, the N-terminus of SAMHD1 governs interactions with Vpx from SIVmnd2 and SIVrcm, but has little effect on Vpx from HIV-2 and SIVmac. Furthermore, we show here that this difference in SAMHD1 recognition is evolutionarily dynamic, with the importance of the N- and C-terminus for interaction of SAMHD1 with Vpx and Vpr toggling during lentiviral evolution. We present a model to explain how the head-to-tail conformation of SAMHD1 proteins favors toggling of the interaction sites by Vpx/Vpr during this virus-host arms race. Such drastic functional divergence within a lentiviral protein highlights a novel plasticity in the evolutionary dynamics of viral antagonists for restriction factors during lentiviral adaptation to its hosts. © 2013 Fregoso et al

The role of visual appearance in Punch’s early-Victorian satires on religion

Satires on various aspects of contemporary religion can frequently be found in the early Victorian editions of Punch. The more strident forms of Protestant evangelicalism, in the earlier 1840s, and Roman Catholic revivalism, around 1850, came in for particular attack. This pattern was partly the result of a drift in the editorial policy of the publication towards a less radical social and political position. However, Catholicism, in both its Roman and Anglican varieties, was particularly vulnerable to the combination of visual and verbal parody employed by Punch because of that denomination’s stress on visual aspects of worship. Evangelicals, by contrast, employed modes of dress and architecture that were similar to those of the secular world of their time and were, thereby, harder to depict as strange and peculiar. The pages of Punch can, therefore, tell us not only about how various Christian groups were viewed in early Victorian England but also about the ways in which they attempted, with varying success, to parry and pre-empt critique in the print media

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages

<p>Abstract</p> <p>Background</p> <p>HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages.</p> <p>Results</p> <p>In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first α-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first α-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.</p> <p>Conclusion</p> <p>These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.</p

Do Voluntary Pollution Reduction Programs (VPRs) Spur Innovation in Environmental Technology

In the context of the EPA's 33/50 program, we study whether a VPR can prompt firms to develop new environmental technologies that yield future emission reduction benefits. Because pollutant reductions generally require costly reformulations of products and/or production processes, environmental over-compliance induced by a VPR may potentially spur environmental innovation that can reduce these costs. Conversely, a VPR may induce a participating firm to divert resources from environmental research to environmental monitoring and compliance activities that yield short-term benefits in reduced emissions. We find evidence that higher rates of 33/50 program participation are associated with significant reductions in the number of successful environmental patent applications four to six years after the program ended; these results suggest a negative relationship between the 33/50 program and longer-run environmental innovation.Research and Development/Tech Change/Emerging Technologies,

The HIV1 Protein Vpr Acts to Enhance Constitutive DCAF1-Dependent UNG2 Turnover

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1)-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1)-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1) complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation