The genetic architecture of DNA replication timing in human pluripotent stem cells

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) – sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

Dynamic innate immune response determines susceptibility to SARS-CoV-2 infection and early replication kinetics

Initial replication of SARS-CoV-2 in the upper respiratory tract is required to establish infection, and the replication level correlates with the likelihood of viral transmission. Here, we examined the role of host innate immune defenses in restricting early SARS-CoV-2 infection using transcriptomics and biomarker-based tracking in serial patient nasopharyngeal samples and experiments with airway epithelial organoids. SARS-CoV-2 initially replicated exponentially, with a doubling time of ∼6 h, and induced interferon-stimulated genes (ISGs) in the upper respiratory tract, which rose with viral replication and peaked just as viral load began to decline. Rhinovirus infection before SARS-CoV-2 exposure accelerated ISG responses and prevented SARS-CoV-2 replication. Conversely, blocking ISG induction during SARS-CoV-2 infection enhanced viral replication from a low infectious dose. These results show that the activity of ISG-mediated defenses at the time of SARS-CoV-2 exposure impacts infection progression and that the heterologous antiviral response induced by a different virus can protect against SARS-CoV-2.

Transcription shapes DNA replication initiation to preserve genome integrity

Background Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. Results We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. Conclusion RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

HIV-1 Gag recruits oligomeric Vpr via two binding sites in p6, but both mature p6 and Vpr are rapidly lost upon target cell entry

The p12 region of MLV Gag and the p6 region of HIV-1 Gag contain late-domains required for virus budding. Additionally, the accessory protein Vpr is recruited into HIV particles via p6. Mature p12 is essential for early viral replication events, but the role of mature p6 in early replication is unknown. Using a proviral vector in which the gag and pol reading frames are uncoupled, we have performed the first alanine-scanning mutagenesis screens across p6, to probe its importance for early HIV-1 replication and to further understand its interaction with Vpr. The infectivity of our mutants suggests that, unlike p12, p6 is not important for early viral replication. Consistent with this, we observed that p6 is rapidly lost upon target cell entry in time-course immunoblotting experiments. By analysing Vpr incorporation in p6 mutant virions, we identified that the 15-FRFG-18 and 41-LXXLF-45 motifs previously identified as putative Vpr-binding sites are important for Vpr recruitment, but that the 34-ELY-36 motif also suggested to be a Vpr-binding site is dispensable. Additionally, disrupting Vpr oligomerization together with removing either binding motif in p6 reduced Vpr incorporation ∼25-50-fold more than inhibiting Vpr oligomerization alone and ∼10-25-fold more than deletion of each p6 motif alone, implying that multivalency/avidity is important for the interaction. Interestingly, using immunoblotting and immunofluorescence, we observed that most of Vpr is lost concomitantly with p6 during infection, but that a small fraction remains associated with the viral capsid for several hours. This has implications for the function of Vpr in early replication. Importance The p12 protein of MLV and the p6 protein of HIV-1 are both supplementary Gag cleavage products that carry proline-rich motifs which facilitate virus budding. Importantly, p12 has also been found to be essential for early viral replication events. However, whilst Vpr, the only accessory protein packaged into HIV-1 virions, is recruited via the p6 region of Gag, the function of both mature p6 and Vpr in early replication is unclear. Here, we have systematically mutated the p6 region of gag and have studied the effects on HIV infectivity and Vpr packaging. We have also investigated what happens to p6 and Vpr during early infection. We show that, unlike p12, mature p6 is not required for early replication and that most of the mature p6, and the Vpr that it recruits, are lost rapidly upon target cell entry. This has implications for the role of Vpr in target cells.

Tree shrew cells transduced with human CD4 and CCR5 support early steps of HIV-1 replication, but viral infectivity is restricted by APOBEC3

The host range of human immunodeficiency virus type 1 (HIV-1) is narrow. Therefore, using ordinary animal models to study HIV-1 replication, pathogenesis, and therapy is impractical. The lack of applicable animal models for HIV-1 research spurred our investigation on whether tree shrews ( Tupaia belangeri chinensis ), which are susceptible to many types of human viruses, can act as an animal model for HIV-1. Here, we report that tree shrew primary cells are refractory to wild-type HIV-1 but support the early replication steps of HIV-1 pseudotyped with the vesicular stomatitis virus glycoprotein envelope (VSV-G), which can bypass entry receptors. The exogenous expression of human CD4 renders the tree shrew cell line infectable to X4-tropic HIV-1 IIIB , suggesting that tree shrew CXCR4 is a functional HIV-1 co-receptor. However, tree shrew cells did not produce infectious HIV-1 progeny virions, even with the human CD4 receptor. Subsequently, we identified tree shrew (ts) apolipoprotein B editing catalytic polypeptide 3 (tsAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity, with virus infectivity reduced 10–1 000-fold. Unlike human APOBEC3G, the tsA3Z2c-Z1b protein was not degraded by the HIV-1 viral infectivity factor (Vif), but markedly restricted HIV-1 replication through mutagenicity and reverse transcription inhibition. The pooled knockout of tsA3Z2c-Z1b partially restored the infectivity of the HIV-1 progeny. This work suggests that tsAPOBEC3 proteins serve as an additional barrier to the development of HIV-1 tree shrew models, even when virus entry is overcome by exogenous expression of human CD4. IMPORTANCE The development of animal models is critical for studying human diseases and their pathogenesis and for evaluating drug and vaccine efficacy. For improved AIDS research, the ideal animal model of HIV-1 infection should be a small laboratory mammal that closely mimics virus replication in humans. Tree shrews exhibit considerable potential as animal models for the study of human diseases and therapeutic responses. Here, we report that human-CD4-expressing tree shrew cells support the early steps of HIV-1 replication and that tree shrew CXCR4 is a functional co-receptor of HIV-1. However, tree shrew cells harbor additional restrictions that lead to the production of HIV-1 virions with low infectivity. Thus, the tsAPOBEC3 proteins are partial barriers to developing tree shrews as an HIV-1 model. Our results provide insight into the genetic basis of HIV inhibition in tree shrews and build a foundation for the establishment of gene-edited tree shrew HIV-1-infected models.

Cell Cycle Regulatory Protein Expression in Multinucleated Giant Cells of Giant Cell Tumor of Bone: do They Proliferate?

Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15INK4b, p16INK4a, p27KIP1, p53 through its effector p21WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.

New SHIVs and Improved Design Strategy for Modeling HIV-1 Transmission, Immunopathogenesis, Prevention and Cure

Simian-human immunodeficiency virus (SHIV) chimeras contain the HIV-1 envelope (env) gene embedded within an SIVmac proviral backbone. Previously, we showed that substitution of Env residue 375-Ser by bulky aromatic residues enhances Env binding to rhesus CD4 and enables primary or transmitted/founder (T/F) HIV-1 Envs to support efficient SHIV replication in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing and analyzing SHIVs containing ten strategically selected primary or T/F HIV-1 Envs corresponding to subtypes A, B, C, AE and AG, each with six allelic variants at position 375. All ten SHIVs bearing wildtype Env375 residues replicated efficiently in human CD4+ T cells, but only one of these replicated efficiently in rhesus CD4+ T cells. This was a SHIV whose subtype AE Env naturally contained a bulky aromatic His residue at position 375. Replacement of wildtype Env375 residues by Trp, Tyr, Phe or His in the other nine SHIVs uniformly led to efficient replication in rhesus CD4+ T in vitro and in RMs in vivo. Env375-Trp – the residue found most frequently among SIV strains infecting Old World monkeys – was favored for SHIV replication in RMs, although some SHIVs preferred Env375-Tyr, -His or -Phe. Nine SHIVs containing optimized Env375 alleles were grown large scale in primary activated rhesus CD4+ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier-2 neutralization sensitivity, transmissible by rectal, vaginal, penile, oral or intravenous inoculation routes, and exhibited acute and early replication kinetics that were indistinguishable from HIV-1 infection in humans. Finally, to expedite future SHIV constructions and eliminate short redundant elements in tat1 and env gp41 that were spontaneously deleted in chronically infected monkeys, we engineered a simplified second-generation SHIV design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary or T/F Envs with bulky aromatic amino acid substitutions at position Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. We further show that SHIV challenge stocks grown in primary rhesus CD4+ T cells are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used effectively to test for vaccine efficacy in preclinical monkey trials.

Systematic analysis of RNAi-accessible SARS-CoV-2 replication steps identifies ORF1 as promising target

A promising approach to combat SARS-CoV-2 constitute small interfering (si)RNAs. We systematically analyzed which SARS-CoV-2 replication steps are accessible for RNAi. We found that siRNAs can target the genome of incoming SARS-CoV-2 terminating replication after cell entry and preventing cytopathy. Suprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting sequences shared by different viral transcripts allowed simultaneous suppression of genomic and subgenomic viral RNAs by a single siRNA. The most effective suppression of viral replication and spread was achieved by siRNAs targeting open reading frame 1 (ORF1) which is solely part of genomic RNA. We propose that an improved accessibility of translational-active ORF1 during early replication, as well as the outcompetition of RNAi factors by common sequences of transcripts are responsible for this. Our work encourages efforts to develop siRNA-based therapies for COVID-19. Targeting ORF1, together with early treatment start or prophylactic use may be key for high antiviral efficiency.

Low replication stress leads to specific replication timing advances associated to chromatin remodelling in cancer cells

ABSTRACTDNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.