In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

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.

Effects of postweaning calorie restriction on accelerated growth and adiponectin in nutritionally programmed microswine offspring.

Poor prenatal development, followed by rapid childhood growth, conveys greater cardiometabolic risk in later life. Microswine offspring exposed to perinatal maternal protein restriction [MPR; "low protein offspring" (LPO)] grow poorly in late-fetal/neonatal stages. After weaning to an ad libitum (AL) diet, LPO-AL exhibit accelerated growth and fat deposition rates with low adiponectin mRNA, despite low-normal body fat and small intra-abdominal adipocytes. We examined effects of caloric restriction (CR) on growth and metabolic status in LPO and normal protein offspring (NPO) randomized to AL or CR diets from weaning. CR transiently reduced growth in both LPO and NPO, delaying recovery in female LPO-CR. Over 7.5-12.5 weeks, linear growth rates in LPO-CR were slower than LPO-AL ( P < 0.001) but exceeded NPO-AL; body weight growth rates fell but were lower in LPO-CR versus NPO-CR. Linear acceleration ceased after 12 weeks. At 16 weeks, percent catch-up in LPO-CR was reduced versus LPO-AL ( P < 0.001). Plasma growth hormone was low in LPO ( P < 0.02). CR normalized fat deposition rate, yet adiponectin mRNA remained low in LPO-CR ( P < 0.001); plasma adiponectin was low in all LPO-AL and in female LPO-CR. Insulin sensitivity improved during CR. We conclude that in LPO: 1) CR delays onset of, but does not abolish, accelerated linear growth, despite low growth hormone; 2) CR yields stunting via delayed onset, plus a finite window for linear growth acceleration; 3) MPR lowers adiponectin mRNA independently of growth, adiposity, or adipocyte size; and 4) MPR reduces circulating adiponectin in LPO-AL and female LPO-CR, potentially enhancing cardiometabolic risk.

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

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.

Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus.

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.

Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection.

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 alpha (polalpha) 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 polalpha and CDK1.

Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance.

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 co-exist as a complex and that FEN1 can regulate telomerase activity at telomeres in mammalian cells.