Biology subjects

McCauley, S. M.

Publications and source records attributed to McCauley, S. M..

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↗

bioRxiv2019

Cyclophilin A protects HIV-1 from restriction by human TRIM5α

The capsid (CA) protein lattice of HIV-1 and other retroviruses encases viral genomic RNA and regulates steps that are essential to retroviral invasion of target cells, including reverse transcription, nuclear trafficking, and integration of viral cDNA into host chromosomal DNA1. Cyclophilin A (CypA), the first cellular protein reported to bind HIV-1 CA2, has interacted with invading lentiviruses related to HIV-1 for millions of years3-7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8-12, but stimulates infectivity in non-human primate cells13-15. Genetic and biochemical data suggest that CypA interaction with CA protects HIV-1 from a restriction factor in human cells16-20. Discovery of the CA-specific restriction factor TRIM521, and of TRIM5-CypA fusion genes that were independently generated at least four times in phylogeny4,5,15,22-25, pointed to human TRIM5 as the CypA-sensitive restriction factor. However, significant HIV-1 restriction by human TRIM521, let alone inhibition of such activity by CypA26, has not been detected. Here, exploiting reverse genetic tools optimized for primary human CD4+ T cells, macrophages, and dendritic cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to restriction by human TRIM5, with the block occurring before reverse transcription. Identical findings were obtained with single-cycle vectors or with replication-competent HIV-1, including sexually-transmitted clones from sub-Saharan Africa. Endogenous TRIM5 was observed to associate with virion cores as they entered the macrophage cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent, but potent inhibition, imposed by human TRIM5. Hopefully this reinvigorates development of CypA-inhibitors for treatment of HIV-1 and other CypA-dependent pathogens27-30.

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↗

bioRxiv2017

Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, these individuals have chronic inflammation associated with heightened risk of cardiovascular pathology. HIV-1 establishes proviruses in long-lived CD4+ memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Though the majority of proviruses that persist during antiviral therapy are defective for production of infectious virions, many are expressed, raising the possibility that the HIV-1provirus or its transcripts contribute to ongoing inflammation. Here we found that the HIV-1 provirus activated innate immune signaling in isolated dendritic cells, macrophages, and CD4+ T cells. Immune activation required transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev was provided in trans, all HIV-1 coding sequences were dispensable for activation except those cis-acting sequences required for replication or splicing. These results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens.

microbiology↗