Rhadinovirus vector-derived human telomerase reverse transcriptase expression in primary T cells

Abstract

The rhadinovirus herpesvirus saimiri (HVS) as a gene delivery vector allows large DNA insertions and long-termed gene expression. In the case of T-cell transduction, such vectors use the viral transformation-associated genes of HVS C488 for T-cell amplification. In this report, we investigated whether the gene for the catalytic telomerase subunit human telomerase reverse transcriptase (hTERT) can substitute for the transformation-associated genes in rhadinoviral T-cell transduction and amplification. By using virus mutants generated by en passant mutagenesis from bacterial artificial chromosomes, we observed a very early and functional transgene expression even by virus mutants without transformation-associated genes. The markers of T-cell transformation by HVS, namely CD2 hyperreactivity, overexpression of interleukin-26, and of the tyrosine kinase Lyn could neither be induced nor enhanced by ectopic hTERT expression. When the viral transformation-associated genes were replaced by the hTERT gene, it was not sufficient for growth transformation, although hTERT was efficiently transduced and functionally expressed by the rhadinovirus vector. Thus, the transformation-associated proteins StpC and Tip are responsible for the T-cell phenotype after transduction by HVS and, additionally, modulate telomerase activity independently of hTERT expression.

Herpesvirus Saimiri Antagonizes Nuclear Domain 10-Instituted Intrinsic Immunity via an ORF3-Mediated Selective Degradation of Cellular Protein Sp100

“…Primary rhesus fibroblasts (kindly provided by A. Kaur, Harvard Medical School, Southborough, MA), OMK cells, and Vero cells were cultivated in Dulbecco's minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum and gentamicin. The HVS strain employed in this study was obtained by reconstitution of infectious viruses using Bac43MOD containing the genomic sequence of HVS strain C488 (10) and a cytomegalovirus (CMV) immediate-early promoter-driven enhanced green fluorescent protein (EGFP) expression cassette inserted in the SwaI site at position 5656, analogous to earlier descriptions (42,47). Stocks of wild-type (wt) and recombinant viruses were prepared, and the amount of viral genomes in the supernatant of infected OMK cells was quantified by quantitative PCR of the MCP locus with primers MCP_for (5=-CCATTTGCCTGTGTTGAGAGTTAA-3=) and MCP_rev (5=-CTCATTACCAGACCCATGTTATGAA-3) and probe MCP_probe (5=/56-FAM/CTCCGAGAG/ZEN/AGCCTATCTGAGATG CCC/3lABkFQ/-3=, where FAM is 6-carboxyfluorescein, ZEN is internal ZEN Quencher, and 3IABKFQ is 3= Ioua Black FQ Quencher) (Integrated DNA Technologies, Coralville, IA).…”

“…Bacterial colonies growing on these plates were further analyzed. Reconstitution of recombinant HVS using purified BAC DNA was performed in OMK cells as described previously (42).…”

The Insulator Protein CTCF Binding Sites in the orf73 /LANA Promoter Region of Herpesvirus Saimiri Are Involved in Conferring Episomal Stability in Latently Infected Human T Cells

“…Transfections were performed when cells were approximately 80% confluent using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The HVS strain employed in this study was obtained by reconstitution of infectious viruses using Bac43MOD containing the genomic sequence of HSV strain C488 analogous to an earlier description (46). Stocks of wild type and recombinant viruses were prepared, and the amount of viral genomes in the supernatant of infected OMKs was determined by quantitative real-time PCR (qPCR) of the major capsid protein (MCP) locus (primers described below).…”

“…Bacterial colonies appearing on these plates were subjected to further characterization by restriction enzyme digest, PCR analyses, and direct sequencing of recombined junctions (see below). Reconstitution of recombinant HVS using purified bacmid DNA was performed as described previously (46). Oligonucleotides (Ultramer; IDT) used for homologous recombination in E. coli were as follows: Mut1CBS_for, 5=-TAGATGGCGATATACGTTCTT TCAAAAAACTATGCAATGATTGGTACCTCAGTACCTAATATAGGC ATGAAACATAACATGGATGACGACGATAAGTAGGG-3=; Mut1CBS_ rev, 5=-CTGTGACCAACTTGTAAAAAATGTTATGTTTCATGCCTAT ATTAGGTACTGAGGTACCAATCATTGCATAGTTTTTTGAACAACC AATTAACCAATTCTGATTAG-3=; Mut2CBS_for, 5=-TTCGTTTGAGC ACCATCTATAATTGCAACAAACACGTTATTTGGTCGACGTTCGATA TACGTTCTTTCAAAAGGATGACGACGATAAGTAGGG-3=; Mut2CBS_ rev, 5=-TGGCCAATCATTGCATAGTTTTTTGAAAGAACGTATATCG AACGTCGACCAAATAACGTGTTTGTTGCAATTCAACCAATTAACCA ATTCTGATTAG-3=; Mut1/2CBS_for, 5=-GGTCGACGTTCGATATAC GTTCTTTCAAAAAACTATGCAATGATTGGTACCTCAGTACCTAAT ATAGGCATGAAACATAACATGGATGACGACGATAAGTAGGG-3=; Mut1/2CBS_rev, 5=-CTGTGACCAACTTGTAAAAAATGTTATGTTTC ATGCCTATATTAGGTACTGAGGTACCAATCATTGCATAGTTTTTT GAACAACCAATTAACCAATTCTGATTAG-3=; delCBS_for, 5=-TTCG TTTGAGCACCATCTATAATTGCAACAAACACGTTATCTAATATAG GCATGAAACATAGGATGACGACGATAAGTAGGG-3=; delCBS_rev, 5=-ACCAACTTGTAAAAAATGTTATGTTTCATGCCTATATTAGATA ACGTGTTTGTTGCAATTCAACCAATTAACCAATTCTGATTAG-3=; Mut1CBS_Rev_for, 5=-TAGATGGCGATATACGTTCTTTCAAAAAAC TATGCAATGATTGGCCACTAGGTGGCTAATATAGGCATGAAACA TAACATGGATGACGACGATAAGTAGGG-3=; Mut1CBS_Rev_rev, 5=-CTGTGACCAACTTGTAAAAAATGTTATGTTTCATGCCTATATTAGC CACCTAGTGGCCAATCATTGCATAGTTTTTTGAACAACCAATTAAC CAATTCTGATTAG-3=; Mut2CBS_Rev_for, 5=-TTCGTTTGAGCACCA TCTATAATTGCAACAAACACGTTATACACTAGATGGCGATATACGT TCTTTCAAAAGGATGACGACGATAAGTAGGG-3=; Mut2CBS_Rev_rev, 5=-TGGCCAATCATTGCATAGTTTTTTGAAAGAACGTATATCGCCA TCTAGTGTATAACGTGTTTGTTGCAATTCAACCAATTAACCAATT CTGATTAG-3=; Mut1/2CBS/delCBS_Rev_for, 5=-TTTCTTCGTTTGAG CACCATCTATAATTGCAACAAACACGTTATACACTAGATGGCGAT ATACGTTCTTTCAAAAAACTATGCAATGATTGGCCGGATGACGA CGATAAGTAGGG-3=; Mut1/2CBS/delCBS_Rev_rev, 5=-CTGTGACCA ACTTGTAAAAAATGTTATGTTTCATGCCTATATTAGCCACCTAGTG GCCAATCATTGCATAGTTTTTTGAAAGAACGTATATCGCCAACCA ATTAACCAATTCTGATTAG-3=.…”

Bacterial Genetics of Large Mammalian DNA Viruses: Bacterial Artificial Chromosomes as a Prerequisite for Efficiently Studying Viral DNA Replication and Functions

“…Also for KSHV, several BACs were constructed and applied for functional analyses (Delecluse et al, 2001;Fan et al, 2006;Lu et al, 2010;Lukac et al, 2001;Luna et al, 2004;Majerciak et al, 2007;Xu et al, 2005Xu et al, , 2006Yakushko et al, 2011Zhou et al, 2002. Additionally, the major rhadinovirus animal model viruses were cloned in BACs, such as RRV (Estep et al, 2007;Zhou et al, 2010), MHV-68 (Adler et al, 2000;Pavlova et al, 2003), and herpesvirus saimiri (HVS;Calderwood et al, 2005;Toptan et al, 2010;White et al, 2003White et al, , 2007. Besides the herpesviruses, BAC-cloning has been successfully applied for the poxvirus Vaccinia virus (Cottingham et al, 2008;Domi & Moss, 2002;Meissinger-Henschel et al, 2011) and cowpox virus (Roth et al, 2011).…”