A(H5N1) Virus Evolution in South East Asia

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in South East Asia. H5N1 is now endemic in poultry in many countries, and represents a major pandemic threat. Here, we describe the evolution of H5N1 virus in South East Asia, the reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. The virus has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The constant surveillance of these changes is of primary importance in the global efforts of the scientific community

TOX4 and NOVA1 Proteins Are Partners of the LEDGF PWWP Domain and Affect HIV-1 Replication

International audiencePWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation

The Integrase Cofactor LEDGF/p75 Associates with Iws1 and Spt6 for Postintegration Silencing of HIV-1 Gene Expression in Latently Infected Cells

International audienceThe persistence of a latent reservoir containing transcriptionally silent, but replication-competent, integrated provirus is a serious challenge to HIV eradication. HIV integration is under the control of LEDGF/p75, the cellular cofactor of viral integrase. Investigating possible postintegration roles for LEDGF/p75, we find that LEDGF/p75 represses HIV expression in latently infected cells. LEDGF/p75 associated with two proteins involved in the control of gene expression and chromatin structure, Spt6 and Iws1, to form a stable complex. Iws1 plays a role in the establishment of latent infection, whereas Spt6 functions to recruit Iws1 and LEDGF/p75 to the silenced provirus and maintains histone occupancy at the HIV promoter. In latently infected cells, depletion of the complex results in reactivation of HIV expression Altogether, our results indicate that a complex containing LEDGF/p75, Iws1, and Spt6 participates in regulating postintegration steps of HIV latency

Use of a multiplex PCR/RT-PCR approach to assess the viral causes of influenza-like illnesses in Cambodia during three consecutive dry seasons.

National audienceAcute respiratory infections are a major cause of mortality and morbidity worldwide. Using multiplex PCR/RT-PCR methods for the detection of 18 respiratory viruses, the circulation of those viruses during 3 consecutive dry seasons in Cambodia was described. Among 234 patients who presented with influenza-like illness, 35.5% were positive for at least one virus. Rhinoviruses (43.4%), parainfluenza (31.3%) viruses and coronaviruses (21.7%) were the most frequently detected viruses. Influenza A virus, parainfluenza virus 4 and SARS-coronavirus were not detected during the study period. Ninety apparently healthy individuals were included as controls and 10% of these samples tested positive for one or more respiratory viruses. No significant differences were observed in frequency and in virus copy numbers for rhinovirus detection between symptomatic and asymptomatic groups. This study raises questions about the significance of the detection of some respiratory viruses, especially using highly sensitive methods, given their presence in apparently healthy individuals. The link between the presence of the virus and the origin of the illness is therefore unclear

Use of a multiplex PCR/RT-PCR approach to assess the viral causes of influenza-like illnesses in Cambodia during three consecutive dry seasons.

National audienceAcute respiratory infections are a major cause of mortality and morbidity worldwide. Using multiplex PCR/RT-PCR methods for the detection of 18 respiratory viruses, the circulation of those viruses during 3 consecutive dry seasons in Cambodia was described. Among 234 patients who presented with influenza-like illness, 35.5% were positive for at least one virus. Rhinoviruses (43.4%), parainfluenza (31.3%) viruses and coronaviruses (21.7%) were the most frequently detected viruses. Influenza A virus, parainfluenza virus 4 and SARS-coronavirus were not detected during the study period. Ninety apparently healthy individuals were included as controls and 10% of these samples tested positive for one or more respiratory viruses. No significant differences were observed in frequency and in virus copy numbers for rhinovirus detection between symptomatic and asymptomatic groups. This study raises questions about the significance of the detection of some respiratory viruses, especially using highly sensitive methods, given their presence in apparently healthy individuals. The link between the presence of the virus and the origin of the illness is therefore unclear

Interaction Of Tox4 And Nova1 Pirs With Ledgf Pwwp By Gst Pull-Down.

<p>GST pull down were performed using purified GST-PWWP protein and Flag-TOX4 PIR or Flag-NOVA1 PIR expressed and present in 293T cells extracts (A and B) or with His-TOX4 PIR or Flag-NOVA1 PIR expressed in <i>E coli</i> and purified (C). Eluted proteins following pull down were separated through 10% PA SDS-PAGE and analyzed by western blot using M2 anti-Flag antibody (A and B), H1029 anti-His antibody (C) and 4C10 anti-GST antibody (A to C). Purified GST was used as negative control for each experiment. B) Effect of DNA and RNA on interaction with PIRs in extracts was studied by DNAse or RNAse treatment of these extracts. C) Effect of DNA or PN on interaction with purified PIRs was studied by addition of a 2.6 kbp 5SG5E4 DNA fragment or a polynucleosome (PN) asssembled on it.</p

Interaction Of Tox4 And Nova1, Full Length Or Pir, With Ledgf/P75 By Co-Immunoprecipitation.

<p>Total extracts of cells transiently expressing HA-LEDGF and either 3×Flag-TOX4, 3xFlag-NOVA1, PIR or full length, Flag-HIV Integrase or Flag-Brd4 were immunoprecipitated with anti-Flag M2 coupled agarose beads. Immunoprecipitated proteins were separated on 10% or 7.5% PA-SDS gels and revealed by immunoblot using antibodies indicated on the left side of the panels and more precisely described in Material and Methods section. A) HA-tagged LEDGF co-immunoprecipitates with 3×Flag tagged full-length and PIR constructs of TOX4 and NOVA1 but not with Flag Brd4. B) HA-tagged LEDGF co-immunoprecipitates Flag-HIV1 integrase. C) DNAse (but not RNAse) treatment of cell extracts abolishes HA-tagged LEDGF co-IP with 3×Flag tagged PIR of TOX4 and NOVA1. Cell extracts were digested by nothing (lane 1), DNAse (lane 2) or RNAse (lane 3) before the co-IP protocol (IP (n>3))</p

Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.

<p>A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.</p