Telomeric G-overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G-overhang generation at human telomeres is poorly understood. Here, we show that G-overhangs can undergo cell cycle-regulated changes independent of telomerase activity. G-overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C-strand fill-in, whereas the sizes of G-overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C-strands are precisely defined throughout the cell cycle, indicating that C-strand resection is strictly regulated. We demonstrate that C-strand fill-in is mediated by DNA polymerase a (pola) and controlled by cyclin-dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G-overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G-overhangs and an increase in telomeric DNA damage. Our results suggest that G-overhang generation at human telomeres is regulated by multiple tightly controlled processes and C-strand fill-in is under the control of pola and CDK1.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68–1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.

Studies from budding yeast and ciliates have suggested that telomerase extension of telomeres requires the conventional DNA replication machinery, yet little is known about how DNA replication proteins regulate telomerase action in higher eukaryotic cells. Here we investigate the role of one of the DNA replication factors, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. FEN1 is a nuclease that plays an important role in DNA replication, repair, and recombination. We show that FEN1 is in complex with telomerase in vivo via telomeric DNA. We further demonstrate that FEN1 deficiency in mouse embryonic fibroblasts leads to an increase in telomere end-to-end fusions. In cancer cells, FEN1 deficiency induces gradual shortening of telomeres but does not alter the single-stranded G-overhangs. This is, to our knowledge, the first evidence that FEN1 and telomerase physically coexist as a complex and that FEN1 can regulate telomerase activity at telomeres in mammalian cells.Telomeres are distinct DNA-protein structures that protect eukaryotic chromosome ends from degradation and inappropriate recombination or fusions. Maintenance of functional telomeres is essential for long term cell proliferation and stem cell self-renewal. In normal human somatic cells, telomeres progressively shorten each time a cell divides (1). When a subset of telomeres becomes critically short, these short telomeres are recognized as damaged DNA (1-6). Activation of the DNA damage response pathway then induces cellular senescence, impairing cell proliferation (7-9).To counteract telomere shortening, the cells activate a special reverse transcriptase, telomerase, to elongate short telomeres. Telomerase adds telomeric DNA repeats to chromosome ends, thus keeping telomeres functional (10, 11). Indeed, telomerase is up-regulated in cells that need long term proliferation potential such as embryonic stem cells, germline cells, cancer stem cells, activated lymphocytes, and the majority of human cancer cells (12-16). The critical roles of telomerase in tumor proliferation and stem cell behavior underscore the importance of understanding the regulatory mechanisms for telomerase action at telomeres.Telomerase elongation of telomeres is a highly coordinated and tightly regulated process, so that the length of the telomeric repeats is kept within a cell type-specific narrow range from 3 to 20 kb in human cells (17). Telomere homeostasis is maintained by a number of proteins associated with the telomere and/or telomerase. These proteins control the recruitment and accessibility of telomerase to telomeres and regulate telomerase activities at telomeres. Defects in certain proteins, among which are DNA metabolic proteins, have been shown to positively or negatively influence telomere length (18).Several studies in yeast and ciliates have suggested that telomerase-dependent telomere extension is coupled with conventional DNA replication and requires certain DNA replication proteins. For example, inactivation of components of budd...

The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection in vivo. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions in vivo. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection.