Biology subjects

Diehl, W. E.

Publications and source records attributed to Diehl, W. E..

6 recordsLinked to original sources

bioRxiv2020

SARS-CoV-2 Spike protein variant D614G increases infectivity and retains sensitivity to antibodies that target the receptor binding domain

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and cells rendered permissive by ectopic expression of various mammalian ACE2 orthologs. Nonetheless, D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts a critical interprotomer contact and that this dramatically shifts the S protein trimer conformation toward an ACE2-binding and fusion-competent state. Consistent with the more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated. These results indicate that D614G adopts conformations that make virion membrane fusion with the target cell membrane more probable but that D614G retains susceptibility to therapies that disrupt interaction of the SARS-CoV-2 S protein with the ACE2 receptor.

microbiology↗

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↗

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

An emergent Ebola virus nucleoprotein variant influences virion budding, oligomerization, transcription and replication

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measure transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013-2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NPs roles in protein structure, virion budding, and transcription and replication.

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↗