Harnessing traditional Chinese medicine polysaccharides for combatting COVID-19.

With the global spread of COVID-19 posing ongoing challenges to public health systems, there is an ever-increasing demand for effective therapeutics that can mitigate both viral transmission and disease severity. This review surveys the landscape of polysaccharides derived from traditional Chinese medicine, acclaimed for their medicinal properties and potential to contribute to the COVID-19 response. We specifically focus on the capability of these polysaccharides to thwart SARS-CoV-2 entry into host cells, a pivotal step in the viral life cycle that informs transmission and pathogenicity. Moreover, we delve into the concept of trained immunity, an innate immune system feature that polysaccharides may potentiate, offering an avenue for a more moderated yet efficacious immune response against various pathogens, including SARS-CoV-2. Our comprehensive overview aims to bolster understanding of the possible integration of these substances within anti-COVID-19 measures, emphasizing the need for rigorous investigation into their potential applications and underlying mechanisms. The insights provided here strongly support ongoing investigations into the adjunctive use of polysaccharides in the management of COVID-19, with the anticipation that such findings could lead to a deeper appreciation and clearer elucidation of the antiviral potentials inherent in complex Chinese herbal remedies.

The level of expression of HPV16 early transcripts is not associated with the natural history of cervical lesions.

The natural history of cervical cancer is closely linked to that of high-risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high-grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Proximity ligation-based sequencing for the identification of human papillomavirus genomic integration sites in formalin-fixed paraffin embedded oropharyngeal squamous cell carcinomas.

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Unraveling the role of hepatitis B virus DNA integration in B-cell lymphomagenesis.

BACKGROUND: Studies have shown that hepatitis B virus (HBV)-associated B-cell non-Hodgkin lymphoma (NHL) constitutes a unique subgroup with distinct clinical features. It still leaves open the question of whether the integration of HBV DNA into the B-cell genome is a causal mechanism in the development of lymphoma. METHODS: Using the hybridisation capture-based next generation sequencing and RNA sequencing, we characterised the HBV integration pattern in 45 HBV-associated B-cell NHL tumour tissues. RESULTS: A total of 354 HBV integration sites were identified in 13 (28.9%) samples, indicating the relatively low integration frequency in B-cell NHLs. High plasma HBV DNA loads were not associated with the existence of HBV integration. The insertion sites distributed randomly across all the lymphoma genome without any preferential hotspot neither at the chromosomal level nor at the genetic level. Intriguingly, most HBV integrations were nonclonal in B-cell NHLs, implying that they did not confer a survival advantage. Analysis of the paired diagnosis-relapse samples showed the unstable status of HBV integrations during disease progression. Furthermore, transcriptomic analysis revealed the limited biological impact of HBV integration. CONCLUSION: Our study provides an unbiased HBV integration map in B-cell NHLs, revealing the insignificant role of HBV DNA integration in B-cell lymphomagenesis.

Complete genome sequence analysis of Reticuloendotheliosis virus integrated in nonhomologous Avipoxvirus.

Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirus（APV） has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.

Post-Integrational DNA Repair of HIV-1 Is Associated with Activation of the DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets.

Integration of the DNA copy of HIV-1 genome into the cellular genome results in series of damages, repair of which is critical for successful replication of the virus. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate the HIV-1 DNA postintegrational repair, even though integration does not result in DNA double-strand breaks. In this study, we analyzed changes in phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056), and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139), and p53 (pSer15) during the HIV-1 DNA postintegrational repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegrational DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between the double-strand DNA break repair and postintegrational repair of HIV-1 DNA.

Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy.

BACKGROUND: The risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern. METHODS: We reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient. RESULTS: A total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein-Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques. CONCLUSIONS: Our results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.).

Antigen specificities and proviral integration sites differ in HIV-infected cells by timing of antiretroviral treatment initiation.

Despite effective antiretroviral therapy (ART), persons living with HIV harbor reservoirs of persistently infected CD4+ cells, which constitute a barrier to cure. Initiation of ART during acute infection reduces the size of the HIV reservoir, and we hypothesized that in addition, it would favor integration of proviruses in HIV-specific CD4+ T cells, while initiation of ART during chronic HIV infection would favor relatively more proviruses in herpesvirus-specific cells. We further hypothesized that proviruses in acute ART initiators would be integrated into antiviral genes, whereas integration sites (ISs) in chronic ART initiators would favor genes associated with cell proliferation and exhaustion. We found that the HIV DNA distribution across HIV-specific versus herpesvirus-specific CD4+ T cells was as hypothesized. HIV ISs in acute ART initiators were significantly enriched in gene sets controlling lipid metabolism and HIF-1α-mediated hypoxia, both metabolic pathways active in early HIV infection. Persistence of these infected cells during prolonged ART suggests a survival advantage. ISs in chronic ART initiators were enriched in a gene set controlling EZH2 histone methylation, and methylation has been associated with diminished long terminal repeat transcription. These differences that we found in antigen specificities and IS distributions within HIV-infected cells might be leveraged in designing cure strategies tailored to the timing of ART initiation.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy.

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values â€‹< â€‹0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

HPV integration and cervical cancer: a failed evolutionary viral trait.

Countless efforts have been made to eradicate cervical cancer worldwide, including improving disease screening and human papillomavirus (HPV) vaccination programs. Nevertheless, cervical cancer still claims the lives of more than 300 000 women every year. Persistent infections with high-risk HPV genotypes 16 and 18 are the main cause of cancer and may result in HPV integration into the host genome. The central dogma is that HPV integration is an important step in oncogenesis, but in fact, it impedes the virus from replicating and spreading. HPV causing cervical cancer can therefore be perceived as a failed evolutionary viral trait. Here we outline the occurrence and mechanisms of HPV integration and how this process results in oncogenic transformation.

Comprehensive Identification and Characterization of HML-9 Group in Chimpanzee Genome.

Endogenous retroviruses (ERVs) are related to long terminal repeat (LTR) retrotransposons, comprising gene sequences of exogenous retroviruses integrated into the host genome and inherited according to Mendelian law. They are considered to have contributed greatly to the evolution of host genome structure and function. We previously characterized HERV-K HML-9 in the human genome. However, the biological function of this type of element in the genome of the chimpanzee, which is the closest living relative of humans, largely remains elusive. Therefore, the current study aims to characterize HML-9 in the chimpanzee genome and to compare the results with those in the human genome. Firstly, we report the distribution and genetic structural characterization of the 26 proviral elements and 38 solo LTR elements of HML-9 in the chimpanzee genome. The results showed that the distribution of these elements displayed a non-random integration pattern, and only six elements maintained a relatively complete structure. Then, we analyze their phylogeny and reveal that the identified elements all cluster together with HML-9 references and with those identified in the human genome. The HML-9 integration time was estimated based on the 2-LTR approach, and the results showed that HML-9 elements were integrated into the chimpanzee genome between 14 and 36 million years ago and into the human genome between 18 and 49 mya. In addition, conserved motifs, cis-regulatory regions, and enriched PBS sequence features in the chimpanzee genome were predicted based on bioinformatics. The results show that pathways significantly enriched for ERV LTR-regulated genes found in the chimpanzee genome are closely associated with disease development, including neurological and neurodevelopmental psychiatric disorders. In summary, the identification, characterization, and genomics of HML-9 presented here not only contribute to our understanding of the role of ERVs in primate evolution but also to our understanding of their biofunctional significance.

HPV, HBV, and HIV-1 Viral Integration Site Mapping: A Streamlined Workflow from NGS to Genomic Insights of Carcinogenesis.

Viral integration within the host genome plays a pivotal role in carcinogenesis. Various disruptive mechanisms are involved, leading to genomic instability, mutations, and DNA damage. With next-generation sequencing (NGS), we can now precisely identify viral and host genomic breakpoints and chimeric sequences, which are useful for integration site analysis. In this study, we evaluated a commercial hybrid capture NGS panel specifically designed for detecting three key viruses: HPV, HBV, and HIV-1. We also tested workflows for Viral Hybrid Capture (VHC) and Viral Integration Site (VIS) analysis, leveraging customized viral databases in CLC Microbial Genomics. By analyzing sequenced data from virally infected cancer cell lines (including SiHa, HeLa, CaSki, C-33A, DoTc2, 2A3, SCC154 for HPV; 3B2, SNU-182 for HBV; and ACH-2 for HIV-1), we precisely pinpointed viral integration sites. The workflow also highlighted disrupted and neighboring human genes that may play a crucial role in tumor development. Our results included informative virus-host read mappings, genomic breakpoints, and integration circular plots. These visual representations enhance our understanding of the integration process. In conclusion, our seamless end-to-end workflow bridges the gap in understanding viral contributions to cancer development, paving the way for improved diagnostics and treatment strategies.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

HIV-1 Capsid Rapidly Induces Long-Lived CPSF6 Puncta in Non-Dividing Cells, but Similar Puncta Already Exist in Uninfected T-Cells.

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.