Biology subjects

Guney, M. H.

Publications and source records attributed to Guney, M. H..

4 recordsLinked to original sources

bioRxiv2023

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus

Antiretroviral therapy (ART) suppresses HIV-1 viremia and prevents progression to AIDS. Nonetheless, chronic inflammation is a common problem for people living with HIV-1 on ART. One possible cause of inflammation is ongoing transcription from HIV-1 proviruses, whether or not the sequences are competent for replication. Previous work has shown that intron-containing RNA expressed from the HIV-1 provirus in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells, activates type 1 interferon. This activation required HIV-1 rev and was blocked by the XPO1 (CRM1)-inhibitor leptomycin. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with shRNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the IFN-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with non-targetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant inhibitory CARD-deletion or phosphomimetic point mutations, indicates that IFIH1 filament formation, dephosphorylation, and association with MAVS, are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1-knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 was revealed by formaldehyde crosslinking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.

microbiology↗

bioRxiv2021

The HIV-1 capsid core is an opportunistic nuclear import receptor

The movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model to interrogate this process due to the genetic and cell biological assays to score virus nuclear entry in living cells. Although initial studies of HIV-1 infection of nondividing cells focused on karyophilic virion proteins, subsequent work revealed the viral capsid (CA), the chief structural component of the pre-integration complex (PIC), to be a critical determinant in nuclear transport1. In support of this model, HIV-1 interactions with NPCs can be altered through CA mutation2, which makes direct contact with nucleoporins (Nups)3-5. Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was strongly dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Collectively, these findings implicate the HIV-1 CA hexameric lattice that encapsulates the viral genome as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion.

cell biology↗

bioRxiv2019

Influence of different glycoproteins and of the virion core on SERINC5 antiviral activity

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three accessory proteins encoded by diverse retroviruses, HIV-1 Nef, EIAV S2, and MLV Glycogag, each independently disrupt SERINC5 antiviral activity, by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., the vesicular stomatitis glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and M-PMV virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. Infectivity of particles, pseudotyped with HIV-1, amphotropic-MLV, or influenza virus glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. Particles generated by all three cores, and pseudotyped with glycoproteins from either avian leukosis virus-A, human endogenous retrovirus K (HERV-K), ecotropic-MLV, HTLV-1, Measles morbillivirus, lymphocytic choriomeningitis mammarenavirus (LCMV), Marburg virus, Ebola virus, severe acute respiratory syndrome-related coronavirus (SARS-CoV), or VSV, were insensitive to SERINC5. In contrast, particles pseudotyped with M-PMV, RD114, or rabies virus (RABV) glycoproteins were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 by particular glycoproteins did not correlate with reduced SERINC5 incorporation into particles or with the route of viral entry. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.\n\nIMPORTANCEThe importance of SERINC5 for inhibition of retroviruses is underscored by convergent evolution among three non-monophyletic retroviruses, each of which encodes a structurally unrelated SERINC5 inhibitor. One of these retroviruses causes tumors in mice, a second anemia in horses, and a third causes AIDS. SERINC5 is incorporated into retrovirus particles where it blocks entry into target cells, via a mechanism that is dependent on the viral glycoprotein. Here we demonstrate that retroviruses pseudotyped with glycoproteins from several non-retroviruses are also inhibited by SERINC5, suggesting that enveloped viruses other than retroviruses may also be inhibited by SERINC5. Additionally, we found that sensitivity to SERINC5 is determined by the retrovirus core, as well as by the glycoprotein. By better understanding how SERINC5 inhibits viruses we hope to extend fundamental understanding of virus replication and of the native role of SERINC5 in cells, and perhaps to advance the development of new antiviral strategies.

microbiology↗

bioRxiv2018

Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex

Drugs that inhibit HIV-1 replication and prevent progression to AIDS do not eliminate HIV-1 proviruses from the chromosomes of long-lived CD4+ memory T cells. To escape eradication by these antiviral drugs, or by the host immune system, HIV-1 exploits poorly defined host factors that silence provirus transcription. These same factors, though, must be overcome by all retroviruses, including HIV-1 and other primate immunodeficiency viruses, in order to activate provirus transcription and produce new virus. Here we show that Vpx and Vpr, proteins from a wide range of primate immunodeficiency viruses, activate provirus transcription in human CD4+ T cells. Provirus activation required the DCAF1 adaptor that links Vpx and Vpr to the CUL4A/B ubiquitin ligase complex, but did not require degradation of SAMHD1, a well-characterized target of Vpx and Vpr. A loss-of-function screen for transcription silencing factors that mimic the effect of Vpx on provirus silencing identified all components of the Human Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN), and MORC2. Vpx associated with the HUSH complex components and decreased steady-state levels of these proteins in a DCAF-dependent manner. Finally, vpx and FAM208A knockdown accelerated HIV-1 and SIVMAC replication kinetics in CD4+ T cells to a similar extent, and HIV-2 replication required either vpx or FAM208A disruption. These results demonstrate that the HUSH complex restricts transcription of primate immunodeficiency viruses and thereby contributes to provirus latency. To counteract this restriction and activate provirus expression, primate immunodeficiency viruses encode Vpx and Vpr proteins that degrade HUSH complex components.

molecular biology↗