Biology subjects

Yurkovetskiy, L.

Publications and source records attributed to Yurkovetskiy, L..

6 recordsLinked to original sources

bioRxiv2024

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

microbiology↗

bioRxiv2023

Deep generative models predict SARS-CoV-2 Spike infectivity and foreshadow neutralizing antibody escape

Recurrent waves of SARS-CoV-2 infection, driven by the periodic emergence of new viral variants, highlight the need for vaccines and therapeutics that remain effective against future strains. Yet, our ability to proactively evaluate such therapeutics is limited to assessing their effectiveness against previous or circulating variants, which may differ significantly in their antibody escape from future viral evolution. To address this challenge, we develop a deep learning method to predict the effect of mutations on fitness and escape from neutralizing antibodies. We use this model to engineer 83 unique SARS-CoV-2 Spike proteins incorporating novel combinations of up to 46 amino acid changes relative to the ancestral B.1 variant. The designed constructs were infectious and evaded neutralization by nine well-characterized panels of human polyclonal anti-SARS-CoV-2 immune sera (from vaccinated, boosted, bivalent boosted, and breakthrough infection individuals). Designed constructs on contemporary SARS-CoV-2 strains displayed similar levels of antibody neutralization escape and similar antigenic profiles as variants seen subsequently (up to 12 months later) during the COVID-19 pandemic despite differences in exact mutations. Our approach provides targeted datasets of antigenically diverse escape variants for an early evaluation of the protective ability of vaccines and therapeutics to inhibit not only currently circulating but also future variants. This approach is generalizable to other viral pathogens.

immunology↗

bioRxiv2023

S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness

Abstract/SummarySARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/535005v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8b4864org.highwire.dtl.DTLVardef@3daad9org.highwire.dtl.DTLVardef@9ba446org.highwire.dtl.DTLVardef@3e1a08_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIS:D614G is ubiquitous among SARS-CoV-2 B-lineage Spikes and is required for infectivity of the main Variants of Concern C_LIO_LIIn an example of convergent evolution, SARS-CoV-2 A lineage viruses maintained transmission chains in the absence of S:D614G, but were instead dependent upon S:Q613H or S:H655Y C_LIO_LIS:D614G and S:H655Y are both adaptations to the polybasic S1/S2 cleavage site C_LIO_LIIncreased infectivity of S:D614G and S:H655Y is associated with a more open RBD conformation and increased steady-state levels of virion-associated S1 C_LI

microbiology↗

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

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↗