The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication

This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold

Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin

Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remarkably large number of disease-causing mutations in the human lamin A gene. This review provides an up-to-date overview of the functions of nuclear lamins, emphasizing their roles in epigenetics, chromatin organization, DNA replication, transcription, and DNA repair. In addition, we discuss recent evidence supporting the importance of lamins in viral infections

Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation

Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

<div><p>Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, <i>ex vivo</i> expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human <i>Herpesviridae</i> family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.</p></div

FM-MSCs are fully permissive to HCMV infection.

<p>(A) FM-MSCs cells were infected with HCMV (strain TB40) at a moi of 1 pfu/cell and fixed at 24 (<i>top panels</i>), 48 (<i>middle panels</i>) or 96 (<i>bottom panels</i>) h pi before being processed for IIF staining of HCMV temporal class representative antigens. Anti-IE (UL123), -E (UL44) or -L (UL99) specific antibodies were used, as indicated at the left of each row. The bright field (BF), the specific secondary antibody signal (TRITC) and the cell nuclei (DAPI) are shown, with merged images presented in the <i>right panels</i>; (B) SNs from HEL cells or from FM-MSCs infected in parallel were harvested at the indicated times pi and used as inocula to determine infectious progeny virion titers via plaque assays on HEL cells as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071412#s2" target="_blank">Materials and Methods</a> section.</p

FM-MSCs are fully permissive to VZV infection.

<p>(A) HEL or FM-MSCs were infected with VZV at a moi<1 pfu/cell, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071412#s2" target="_blank">Materials and Methods</a> section. At the indicated time pi cells were analyzed by optical microscopy; (B) To determine if the cytopathic effect observed in (A) was due to productive VZV infection, infected cells were harvested at different time points pi along with their SNs, and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071412#s2" target="_blank">Materials and Methods</a> to reinfect naive HEL cells. Infectious virus production was subsequently quantified by means of plaque counting. Each reported pfu value is referred to 10⁵ infected cells.</p

FM-MSCs are not permissive to HHV-8 productive infection.

<p>FM-MSCs (<i>left panels</i>) or HUVECs (<i>right panels</i>) were infected with HHV-8 at low (A, B) or high (C) moi, and nucleic acids were extracted at the indicated times pi. (A) Single step PCR (1st) targeting the HHV-8 genome. (B, C) Single step (1st) or nested (2nd) RT-PCR against the indicated transcripts. k- = mock infected FM-MSC cells. RT- = PCR amplification of RNA samples without RT.</p

FM-MSCs are susceptible to HSV-1 and HSV-2 infection.

<p>(A) FM-MSCs were either mock infected (<i>left panel</i>) or infected with >1 pfu/cell of the indicated viruses before being analyzed with an inverted optical microscope as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071412#s2" target="_blank">Materials and Methods</a> section. The typical cytopathic effect shown is observed starting 12–18 h pi; (B) FM-MSCs were either mock infected (<i>top panels</i>) or infected with >1 pfu/cell of the indicated viruses before being fixed and processed for IFF using a mAb directed against the late glycoprotein gD to detect infected cells, and DAPI to visualize cell nuclei. The bright field (BF) is shown in the <i>left panels</i>, and a merged image of all channels is shown in the <i>right panels</i>. Formation of typical syncytia in infected cells is evident starting at 18 h pi, as shown in the HSV-2 <i>bottom panels</i>; (C) SNs of infected FM-MSCs were collected at the indicated times pi and used to reinfect Vero (for HSV-1) or BHK (for HSV-2) cells to quantify infectious virion yields in the extracellular fluid of FM-MSCs by means of plaque assays as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071412#s2" target="_blank">Materials and Methods</a> section.</p

FM-MSCs are not permissive to EBV productive infection.

<p>FM-MSCs (<i>left panels</i>) or primary B lymphocytes (<i>right panels</i>) were infected with EBV, and nucleic acids were extracted at the indicated times pi. (A) Single step PCR (1st) targeting the EBV genome. (B) Single step (1st) or nested (2nd) RT-PCR targeting the indicated transcripts. k- = mock infected FM-MSC cells. RT- = PCR amplification of RNA samples without RT.</p