Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function

Abstract

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM ؊ lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM ؊ lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM ؊ line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM ؊ cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. T he human cytomegalovirus (HCMV) life cycle involves a complex interplay between the virus and the host, with the virus exploiting the host cellular machinery for many of its own functions and, ultimately, releasing fully infectious virions. During a permissive HCMV infection, after virions have entered the cell, the tegument proteins and virus genome are independently trafficked to the nucleus. In fibroblasts, large bipolar viral replication centers (RCs) are formed within 48 h postinfection (hpi) and certain host cellular proteins become strongly associated with these RCs (1; reviewed in reference 2). These proteins include the regulatory protein p53 (3), as well as numerous components of the host cellular DNA damage response (DDR) and repair pathways (4-8).Many virus infections affect the DDR. The interactions span a range of up-and downregulations and include a complex dynamic between the virus and its host's damage response (as reviewed in references 6 and 9). Some viruses appear to require DDR proteins for efficient replication (10, 11), while for other viruses an efficient DDR can be detrimental to their DNA replication (12-21). Studies from several labs, including our own, have shown that HCMV infection initiates the ataxia telangiectasia mutated (ATM)-dependent double-strand break (DSB) ...

Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP

“…We assessed the levels of endogenous miRNA after HCMV infection and the downstream effects of the binding of miR-200b-3p and miR-200c-3p to the 3′-UTR of UL122 and UL123 [ 34 , 35 ] in the human foreskin fibroblast-1 (HFF-1) cell line that is highly permissive for acute lytic replication. We infected these cells with HCMV Towne strain at a multiplicity of infection (MOI) of 0.1 [ 27 , 81 , 82 , 83 , 84 , 85 ].…”

“…All cell cultures were maintained with penicillin (50 U/mL) and streptomycin (50 μg/mL) in a humidified 5% CO 2 atmosphere at 37 °C. The HFF-1 and U373MG cells were infected with HCMV Towne strain (ATCC, Manassas, VA, USA) at an MOI of 0.1 to establish acute lytic replication [ 27 , 81 , 82 , 83 , 84 ]. We latently infected THP-1 cells with the HCMV Toledo strain (low-passage clinical isolate) at an MOI of 5 [ 28 , 89 , 90 ].…”

The role of Rho family GTPases in human cytomegalovirus infection

“…The HCMV infected cells produce three distinct types of viral particles. The first type contains the abovementioned mature capsids, the non-infectious enveloped particles (NIEP) [21] and finally the non-replicating particles which are termed dense bodies (DB) [22]. The NIEPs comprise from the same envelope proteins that the mature particles have, without the viral genome.…”