HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin

<p>Abstract</p> <p>Background</p> <p>Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity.</p> <p>Results</p> <p>Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD).</p> <p>Conclusions</p> <p>Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.</p

Evolution and Spread of Ebola Virus in Liberia, 2014–2015

The 2013–present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.Organismic and Evolutionary Biolog

Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures

Ebola virus epidemiology, transmission, and evolution during seven months in Sierra Leone

The 2013-2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics

The role of LEDGF/p75 in transcriptional regulation

The Lens Epithelial Derived Growth Factor p75 (LEDGF/p75) is a chromatin bound protein whose cellular function is not yet clearly known. A role in transcriptional regulation had been previously proposed based on its interaction with the basal transcriptional machinery and on its effects on the expression of genes involved in the cellular response to environmental stresses. To further elucidate the function of LEDGF/p75, we conducted a global and unbiased evaluation of the role of this protein in gene expression. To that aim, we performed a microarray analysis of cellular gene expression in cells that are severely depleted of LEDGF/p75. To minimize cell type-specific observations, we used three different LEDGF/p75 deficient human cell lines: embryonic kidney epithelial cells (HEK293) and two different lines of CD4+ T cells (SupT1 and Jurkat). By taking the intersection of the three data sets, using a fold-change of greater than two and a student t-test confidence value of 90% we have identified a group of genes whose expression is dysregulated in at least two of the cell types tested. The potential role of LEDGF/p75 in transcriptional regulation of responsive genes has been described as operating at the level of their promoters as a general co-activator, connecting members of the basal transcriptional apparatus to DNA sequence specific transcriptional activators. However, the observation that LEDGF/p75 is bound to sites within the chromatin across the genome suggests that this protein is also present inside actively transcribed genes. In correlation with this observation, LEDGF/p75 has been demonstrated to promote integration of cDNA copies of the HIV-1 genome within actively transcribed genes. This localization of LEDGF/p75 inside actively transcribed genes suggests a possible role of LEDGF/p75 in transcriptional elongation. To further investigate whether LEDGF/p75 has a role in transcriptional elongation, we performed a transcriptional profile analysis of genes we previously identified as dysregulated in SupT1 and HEK293 cells. We used chromatin immunoprecipitation and quantitative real time PCR analysis to demonstrate that LEDGF/p75 is located on the intragenic regions of these genes as well as their promoters. LEDGF/p75 occupancy on these genomic locations correlated with the gene transcriptional activity. We also demonstrate that LEDGF/p75 coimmunoprecipitates with CDK9 and the FACT complex, two separate components of the transcriptional elongation complex, and corroborated this association by quantitative confocal colocalization. In order to understand the mechanism of LEDGF/p75 on transcriptional regulation at the promoter level, we studied the effect of LEDGF/p75 on the shared bi-directional promoter of the LEDGF/p75-regulated genes DTX3L and PARP9. Our data indicate that LEDGF/p75 negatively regulates the expression of this promoter. Two known motifs within LEDGF/p75, the HIV-1 Integrase binding domain and the PWWP domain, were both required for this inhibitory effect. These data suggest that the effect of LEDGF/p75 on the transcription of the DTX3L/PARP9 genes at the chromatin level may involve not only the promoter regions but other genetic elements as well. Alternatively, LEDGF/p75 could require the interaction with other cellular factors that are limiting in cells that are over expressing LEDGF/p75. LEDGF/p75 has been reported to be involved in the adaptation to cellular stress in cells over expressing this protein; however, our data indicate that under basal endogenous conditions LEDGF/p75 does not significantly affect the regulation of genes involved in stress response. To clarify this potential protective effect we studied the capability of LEDGF/p75-deficient cells to adapt to environmental stresses. Our data indicate a protective effect of LEDGF/p75 to these stresses being necessary for cell viability under these conditions. To further understand this effect of LEDGF/p75 on cellular response to stress we determined the differential distribution of LEDGF/p75-interactor proteins involved in transcriptional regulation in response to cellular insults. We demonstrate that LEDGF/p75 influences the subcellular distribution of SSRP1 and CDK9 in response to environmental stresses. LEDGF/p75 was required to mediate recruitment of both SSRP1 and CDK9 to transcriptionally active complexes following these cellular insults. In summary, our data indicate that LEDGF/p75 interacts with components of the basal transcription machinery and of the transcriptional elongation complex, suggesting that this protein regulates the transcription of LEDGF/p75-responsive genes at the initiation and elongation steps of transcription. Additionally, LEDGF/p75 participates in the recruitment of the transcriptional elongation factors SSRP1 and CDK9 to chromatin under basal conditions and in response to cellular stresses

SUMOylation of the Lens Epithelium-Derived Growth Factor/p75 Attenuates Its Transcriptional Activity on the Heat Shock Protein 27 Promoter

Lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional coactivators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are posttranslationally modified by SUMO (small ubiquitin-like modifier)-1 and SUMO-3. Three SUMOylation sites, K75, K250, and K254, were mapped on the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75, and significantly increased its transcriptional activity on the heat shock protein 27 promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate heat shock protein 27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress

Qualitative Profiling of the Humoral Immune Response Elicited by rVSV-ΔG-EBOV-GP Using a Systems Serology Assay, Domain Programmable Arrays

Development of an effective vaccine became a worldwide priority after the devastating 2013–2016 Ebola disease outbreak. To qualitatively profile the humoral response against advanced filovirus vaccine candidates, we developed Domain Programmable Arrays (DPA), a systems serology platform to identify epitopes targeted after vaccination or filovirus infection. We optimized the assay using a panel of well-characterized monoclonal antibodies. After optimization, we utilized the system to longitudinally characterize the immunoglobulin (Ig) isotype-specific responses in non-human primates vaccinated with rVSV-ΔG-EBOV-glycoprotein (GP). Strikingly, we observed that, although the IgM response was directed against epitopes over the whole GP, the IgG and IgA responses were almost exclusively directed against the mucin-like domain (MLD) of the glycan cap. Further research will be needed to characterize this possible biased IgG and IgA response toward the MLD, but the results corroborate that DPA is a valuable tool to qualitatively measure the humoral response after vaccination