JVI Accepts, published online ahead of print on 1 February 2012 J. Virol. doi:10.1128/JVI.06995-11 Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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Quantification and Analysis of Thymidine Kinase Expression

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from Acyclovir-Resistant G-String Insertion and Deletion Mutants

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in Herpes Simplex Virus-Infected Cells

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Dongli Pan and Donald M. Coen*

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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical

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School, 250 Longwood Avenue, Boston, MA 02115

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Running title: TK in ACVr mutant-infected cells

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*Corresponding author. Mailing address: Department of Biological Chemistry and

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Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA

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02115. Phone: (617) 432-1691. Fax: (617) 432-3833.

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Email: [email protected]

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Abstract

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To be clinically relevant, drug resistant mutants must both evade drug action and retain

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pathogenicity. Many acyclovir resistant herpes simplex virus mutants from clinical

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isolates have one or two base insertions (G8 and G9) or one base deletions (G6) in a

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homopolymeric run of seven guanines (G-string) in the thymidine kinase (tk) gene.

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Nevertheless, G8 and G9 mutants express detectable TK activity, and can reactivate from

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latency in mice, a pathogenicity marker. Based on studies using cell-free systems,

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ribosomal frameshifting can explain this ability to express TK. To investigate

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frameshifting in infected cells, we constructed viruses that express epitope tagged

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versions of WT and mutant TKs. We measured TK activity by plaque autoradiography

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and expression of frameshifted and unframeshifted TK polypeptides using a very

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sensitive immunoprecipitation-Western blot method. The G6 mutant expressed ~0.01%

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of wild type levels of TK polypeptide. For the G9 mutant, consistent with previous

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results, much TK expression could be ascribed to reversion. For the G8 mutant, from

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these assays and pulse labeling studies, we determined the ratio of synthesis of

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frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons

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before or after the G-string argue that frameshifting can initiate within the first six

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guanines. However, frameshifting efficiency was altered by stop codons downstream of

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the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild type level of

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full-length TK, considerably lower than estimated previously. Thus, remarkably low

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levels of TK are sufficient for reactivation from latency in mice.

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Introduction Resistance of herpes simplex virus (HSV) to acyclovir (ACV) and the related more

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orally available drugs, valacyclovir and famciclovir, is a critical concern as it occurs in 5-

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10% of immunocompromised patients and 15-30% of bone marrow transplant patients

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with HSV disease (9, 23, 28). To confer clinically significant drug resistance, a viral

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mutant must both evade drug action and retain pathogenicity. The vast majority of ACV

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resistant (ACVr) clinical isolates have mutations in the viral tk gene (2), which encodes

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the enzyme required to phosphorylate and thus activate the drug in infected cells.

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Although ACVr mutations can arise in many different sites in the tk gene, about half of

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the ACVr tk mutants from clinical isolates have insertions or deletions at homopolymeric

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stretches of Cs and Gs (26). Of these, about half have an insertion of one or two Gs (G8

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or G9), and 5% have a deletion of one G (G6) in a run of 7Gs (G-string) (Fig. 1) (2, 5, 11,

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27). These mutants should produce polypeptides with no TK activity since the sequences

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downstream of the insertion or deletion are out of frame. However, G8 and G9 mutants

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are not truly TK-negative (reviewed in (13)). Both G8 and G9 exhibit low, but higher-

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than-background TK activities as seen by plaque autoradiography (14, 16, 17), which can

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suffice to permit some reactivation of the viruses from latent infection in mice, a marker

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of pathogenicity (14). In contrast, isogenic truly TK-negative viruses fail to reactivate

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from latency (4, 7, 10, 29, 30). To our knowledge, no one has characterized the TK

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activity or polypeptide expression of G6 mutants.

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In the case of G9 mutants, plaque autoradiography studies show that much of their

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TK activity and ability to reactivate from latency results from reversion to a TK-positive

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phenotype, often by addition of a tenth G (12, 16). Reversion can also account for some

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of the ability of G8 mutants to reactivate from latency (14, 25, 26). Nevertheless, there is

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considerable evidence that much of the TK activity of G8 mutants is a result of ribosomal

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frameshifting (12, 16, 20). Plaque autoradiography provides no evidence for reversion of

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G8 mutants in cell culture (14, 17). Full-length TK expression has been observed in G8

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mutant-infected cells, and has been recapitulated in an in vitro rabbit reticulocyte lysate

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translation system, where it was shown to be due to translational rather than other

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mechanisms (20). In vitro, the frequency of frameshifting has been estimated at ~1% (19,

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20). The mechanism of ribosomal frameshifting on the G8 sequence in reticulocyte

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lysates has been studied using a dual-reporter system, and found to be unusual, involving

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the G-richness of the frameshifting signal rather than slippage and pausing (19).

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However, frameshifting has not been quantified nor its mechanisms explored in G8-

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mutant infected cells.

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To investigate TK expression in G-string insertion and deletion mutant-infected

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cells, we constructed viruses expressing epitope-tagged TK, which allowed us to very

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sensitively detect and quantify the amount of full-length and truncated TK polypeptides

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arising from mutant viruses, and thus assess frameshifting efficiency of the G8 virus. We

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also used this system to address certain mechanistic questions about frameshifting in

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infected cells. Finally, previous measurements of TK expression from G8 and G9

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mutants have been indirect, relying on plaque autoradiography assays in which TK

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activity was compared to that of mutants with varying tk mRNA levels (6, 8, 17, 21).

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Our assays permitted us to measure the amount of TK protein expressed by G8, G9, and

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G6 mutants directly, and have led us to considerably revise downward estimates of the

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amount of TK that is sufficient to permit reactivation from latency in a mouse model.

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Materials and Methods

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Cells and viruses. African green monkey kidney (Vero) and TK- human osteosarcoma

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(143B) cells were maintained in Dulbecco’s modified Eagle’s media, supplemented with

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10% newborn calf serum and 10% fetal bovine serum, respectively, at 37 °C and 5%

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CO2. A bacterial artificial chromosome (BAC) was developed containing wild-type (WT)

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HSV-1 strain KOS in which Cre expressing vector sequences flanked by a pair of loxP

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sites are deleted from the viral genome by Cre recombinase upon transfection into cells,

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leaving a 36 bp in-frame sequence (34bp loxP+2bp) ~ 380 bp downstream of the G-string

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within sequences encoding a loop on the surface of the TK structure. The insertion does

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not affect TK activity. A more detailed description of the BAC will be presented in a

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separate paper (I. Jurak et al., manuscript in preparation). Mutations were introduced into

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the BAC using two-step Red-mediated recombination (31) with a kanamycin resistant

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gene as a positive selection marker. Briefly, mutations were introduced together with the

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selection marker, which was later removed by I-SceI cleavage and intramolecular Red

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recombination utilizing previously introduced sequence duplication, leaving no additional

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sequences besides the mutations. The primers used were listed in Supplemental Table 1,

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which can be found at https://coen.med.harvard.edu. The integrity of BAC DNAs was

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verified by restriction digestion, and the presence of the engineered mutations and the

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absence of unwanted mutations in the tk gene were verified by sequencing the tk region

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both in BACs and in the viruses derived from them. The untagged G8 mutant was

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described in (14).

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Plaque autoradiography. Plaque autoradiography was performed by modifying a

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previously described protocol (14). The modifications were that the dishes were seeded

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with 4x105 143B cells 24 h prior to addition of a inoculum of 200 PFU of virus/dish and

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that radiolabeling was carried out for 20 h in the presence of 5 µCi/ml of [3H]thymidine.

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Immunoprecipitation (IP) 143B cells were cultured in 100-mm-diameter dishes at 107

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cells/dish, infected at an MOI of 10 and incubated at 37 °C for the times indicated.

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Monolayers were washed with phosphate buffered saline (PBS), scraped into 0.5 ml of

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lysis buffer (50 mM HEPES-KOH [pH 7.4], 1% Triton X-100, 150 mM NaCl, 10%

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glycerol, 0.1 mM DTT, and 2 mM EDTA plus one Complete EDTA-free protease

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inhibitor tablet [Roche] per 50 ml), and rocked at 4 °C for 1 h before being centrifuged.

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After setting aside an aliquot for subsequent Western blot analysis, the supernatant was

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incubated with 20 μl of settled EZ-view anti-FLAG M2 affinity resin (Sigma) at 4 °C

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with rotation for 4 h. The beads were then washed twice by rotating in 800 μl of lysis

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buffer for 20 min at 4 °C before being incubated in 30 μl of 2x SDS-PAGE sample buffer

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(62.5 mM Tris-HCl [pH6.8], 25% glycerol, 2% SDS, 0.01% bromophenol blue, and 5%

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β-mercaptoethanol) at 95 °C for 5 min to elute proteins.

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Western blot analyses. Total proteins from infected cells, prepared by lysing cells in

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each well of a 12 well plate with 100 µl of SDS-PAGE sample buffer; aliquots of lysates

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used for IPs, prepared by mixing with equal volumes of 2x SDS-PAGE sample buffer,

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and samples eluted from affinity resin following IP were resolved on 12 % SDS-

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polyacrylamide gels. Proteins from gels were transferred to polyvinylidene fluoride

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membranes (Pall Corporation) using a Trans-blot semi-dry transfer cell (Bio-Rad). For

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probing the blots, the following antibodies were used: anti-FLAG antibody conjugated to

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horseradish peroxidase (Sigma) at a dilution of 1:1,000; anti-TK rabbit polyclonal

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antibody (generously provided by Bill Summers, Yale University) at 1:1,000; anti-HA

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rabbit polyclonal antibody (Sigma) at 1:1,000; anti-ICP8 rabbit polyclonal antibody (a

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kind gift of David Knipe, Harvard University) at 1:10,000; and to detect bound rabbit

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antibodies, goat anti-rabbit IgG conjugated to horseradish peroxidase (Southern biotech)

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at 1:2,000. Signals were visualized by applying ECL reagents (Pierce) and exposing the

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membranes to films. Western blot images were quantified by densitometry using

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Quantity One version 4.5 (Bio-Rad).

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Northern blot hybridization. 143B cells in 6-well plates were infected at an MOI of 10,

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and incubated for 5h at 37°C. RNAs were extracted using an RNeasy Mini Kit (Qiagen)

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and separated on a 1% agarose gel in NorthernMax-Gly running buffer (Ambion).

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Ethidium bromide staining showed that the same amount of RNA was loaded in each

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well (data not shown). The gel was transferred to a Nytran nylon membrane (Whatman)

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using a Turboblotter kit (Whatman) following the manufacturer’s instructions, ultraviolet

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crosslinked, and hybridized with a tk riboprobe, and washed using buffers provided in the

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NorthernMax-Gly kit (Ambion) following the manufacturer’s instructions. Following

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obtaining the image using a phosphorimager, the membrane was then hybridized with an

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ICP0 probe without stripping. After visualizing that image the same membrane was

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hybridized with an actin probe without stripping and visualized. The tk, ICP0 and actin

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riboprobes were generated by in vitro transcription of linearized pTKprobe, pICP0probe

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and pActinprobe plasmids, respectively. pTKprobe was generated by inserting a 251 bp

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tk gene fragment (47395-47646 based on NC_001806 [NCBI] ) into a pSL301 vector

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(Invitrogen); pICP0proble was generated by inserting a 333 bp BamHI-XhoI ICP0 gene

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fragment from pcDNAICP0 into pSL301 ( pcDNAICP0 is a plasmid containing the

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spliced cDNA of ICP0 transcripts inserted into EcoRI-XbaI sites of

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pcDNA3.1[Invitrogen]); pActinprobe was generated by inserting a 140 bp fragment of

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the exon 4 of the human ß–actin gene (nucleotides 682-821 based on NM_001101

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[NCBI]) into pIDTBblue (Integrated DNA Technologies).

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Pulse-chase protocol. Infections were performed as described above for IP in 100-mm-

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diameter dishes. Cells were incubated for 5h at 37°C before the media were replaced

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with 2 ml/dish fresh media containing 100 µCi/ml [35S]methionine (Perkin Elmer). Thirty

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min later, the radioactive media were removed, and the monolayers were washed twice

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with PBS and supplied with fresh media (which contains unlabeled methionine at 30

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mg/l). At the time points indicated, the monolayers were washed twice with PBS and

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scraped into 0.6 ml of lysis buffer and lysed at 4 °C for 1 h. A portion of the cleared

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lysate was set aside, and the rest was used for IP following the above procedure except

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that EZ-view anti-HA affinity gel (Sigma) was used, and beads were washed 5 times

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instead of 2 times. IP and lysate samples were resolved on 12% SDS-polyacrylamide

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gels. Gels were then dried and exposed to a phosphorimager screen, and the band

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intensities were quantified by densitometry using Quantity One version 4.5 (Bio-Rad)

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and corrected for different methionine contents (full-length TK, 13 methionines;

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truncated G8 TK, 7 methionines).

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Results

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Levels of full-length TK polypeptides in G-string mutant infected cells. We wished to

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analyze the expression of full-length TK by G8 and G9 mutant viruses in infected cells,

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and also investigate whether G6 viruses express TK activity and full-length TK. These

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studies required a sensitive and quantitative assay of TK polypeptide. Although we had

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previously observed radiolabeled full-length TK from G8 infected cells using

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immunoprecipitation (IP) with an anti-TK antibody (20), the sensitivity was too low for

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the assay to be quantitative. Our initial attempts using Western blotting with a

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commercially available anti-TK antibody also failed due to insufficient sensitivity (data

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not shown). To more quantitatively detect low levels of TK from the mutants, we

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constructed viruses in which wild type (WT) HSV-1 and G8, G9, and G6 mutant TKs

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included a FLAG tag at the C-terminus of the TK open reading frame (Fig. 1), using a

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bacterial artificial chromosome (BAC) derived from WT HSV-1 strain KOS (I. Jurak et

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al., manuscript in preparation). Addition of the FLAG tag did not affect virus replication,

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TK polypeptide expression as monitored by comparing WT and WT-FLAG using

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Western blotting (data not shown), or enzyme activity as monitored by comparing G8 and

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G8-FLAG using plaque autoradiography (Fig. 2A). As had been seen previously with

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untagged G9 mutants (16, 17), plaque autoradiography revealed a heterogeneous

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population from G9-FLAG with some plaques exhibiting high levels of TK activity and

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some low (Fig. 2A). This variability is consistent with a high frequency of phenotypic

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reversion for this mutant with reversion occurring at different times during plaque

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formation, as has been confirmed with untagged G9 mutants (16, 17). In contrast, G8-

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FLAG formed plaques with similar TK activities (Fig. 2A). G6-FLAG formed plaques

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without detectable TK activity (Fig. 2A).

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To detect full-length TK expression in infected cells, we optimized a procedure in

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which FLAG-tagged TK polypeptide was efficiently immunoprecipitated with anti-

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FLAG antibody (~99% of FLAG-TK immunoprecipitated (not shown), and was then

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detected by Western blot, also with anti-FLAG antibody. The optimized procedure,

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denoted IP-Western (FLAG), was sensitive enough to detect 0.01% of the WT level of

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TK polypeptide, based on a dilution series in which the WT-FLAG infected lysate was

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diluted in mock infected cell lysate prior to IP (Fig. 2B, left panel). The assay revealed

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that G8-FLAG, G9-FLAG, and G6-FLAG express about 0.1%, 0.5%, and 0.01% of the

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WT level of full-length TK, respectively. The detection of TK from G6-FLAG was

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reproducible, and was not due to contamination from adjacent samples, because when the

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G6-FLAG sample was electrophoresed adjacent to a sample from mock-infected cells,

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full-length TK was still clearly detectable (Fig. 2B, right panel). Nevertheless, the

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undetectable TK activity of G6-FLAG in plaque autoradiography correlated with a

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~10,000-fold decrease in full length TK expression.

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For G8-FLAG and G9-FLAG, similar results were obtained in three additional

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independent experiments in which WT-FLAG samples were diluted after IP. In all

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experiments, full-length TK levels for G8-FLAG and G9-FLAG were in the range of

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0.08-0.15% and 0.3-1%, respectively. The latter range of values is consistent with

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amounts of TK enzyme activity measured in lysates of cells infected with a different G9

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mutant (12).

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We also measured TK polypeptide expression using a polyclonal TK antibody

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generously provided by Bill Summers (Yale University), and a direct Western blot

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procedure. Based on a dilution series made by diluting cell lysates in SDS-PAGE sample

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buffer, we detected about 0.3% of the WT level of full-length TK from G9-FLAG, and

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about 0.1% from G8-FLAG (and from an untagged G8 virus (data not shown)), and

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background levels for G6-FLAG (Fig. 2C), consistent with our results using IP-Western

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(FLAG). This Western blot also revealed bands from N-terminal TK products with the

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expected mobilities (Fig. 1). These products appear to be less abundant than full length

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TK from WT-FLAG due to containing only a portion of the epitopes recognized by the

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polyclonal antibody, and to reduced synthesis (see below). A similar, relatively low

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abundance of N-terminal products was also observed with untagged G8 and G9 viruses

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that were not derived from the BAC (Supplemental Figure 1 at

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https://coen.med.harvard.edu and data not shown). The direct Western blot procedure

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also allowed us to measure ICP8 expression as an internal control for infection and

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loading. All viruses expressed similar amounts of ICP8 (Fig. 2C). Thus, using both the

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IP-Western (FLAG) and direct Western blot methods, the G8 mutant virus expressed

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~0.1% of WT levels of full-length TK polypeptide. These levels are 5- to 10-fold lower

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than previous estimates of TK expression using less direct methods ((3, 15-18); see

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Discussion).

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Does reversion account for TK expression from G8 and G9 mutants? Our plaque

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autoradiography results suggested that the G9 mutant reverted to high level TK

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expression while TK expression by the G8 mutant was more stable. To examine this

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further, we purified single plaques from G9-FLAG or G8-FLAG infected Vero cells,

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amplified them, and used the amplified stock to infect 143B cells. Stocks derived from

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individual plaques of G9-FLAG showed great variation in the levels of expression of full-

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length TK polypeptide shown by IP-Western (FLAG) (0.3-3% in the five plaques

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selected) (Fig. 3), consistent with phenotypic reversion occurring during plaque

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formation resulting in mixed populations of virus. This variation was not due to

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differences in infection, as the lysates used for IP-Western (FLAG) expressed similar

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levels of ICP8 (Fig. 3). In contrast, stocks derived from five different G8-FLAG plaques

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showed uniform full-length TK expression (Fig. 3), supporting the notion that reversion

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plays at most a limited role in the expression of active TK by G8. This result is

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consistent with our never having observed high levels of TK activity in G8 plaques in

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assays of thousands of such plaques ((3, 14-17, 20) and data not shown).

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Frameshifting efficiency in G8 mutant infected cells. We next wished to determine the

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frameshifting efficiency for the G8 mutant, which entailed measuring the ratio of the full-

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length frameshifted product to the N-terminal unframeshifted product. To this end, an

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HA-tag was introduced near the N-terminus of TK, which was already FLAG-tagged at

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its C-terminus, in viruses containing either the WT or the G8 mutant tk gene. The tag was

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not added at the very N-terminus of TK to avoid perturbation of the important viral gene

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UL24, which overlaps tk for 138 base pairs (22). The added HA tag did not affect TK

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activity, as shown by plaque autoradiography (data not shown). After cells were infected

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with HA-WT-FLAG and HA-G8-FLAG viruses, the levels of the N-terminal and full-

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length proteins were determined by reference to a dilution series in Western blot (HA)

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and IP-Western (FLAG) assays (Fig. 4A, B). The full-length protein in HA-G8-FLAG

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infected cells was about 0.1% of that in HA-WT-FLAG infected cells, the same value we

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had obtained using viruses with FLAG tags alone (Fig. 2B), suggesting the added HA tag

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did not affect TK expression. Interestingly, the N-terminal product expressed by HA-G8-

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FLAG was present at only about 10% the level of full-length TK from HA-WT-FLAG, as

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judged by comparison with a dilution series (Fig. 4B). In contrast, tk mRNA produced by

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the mutant was reduced less than 2-fold, as assayed by Northen blot hybridization and

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densitometric scanning (Fig. 4C).

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To test whether the reduced level of N-terminal product was due to reduced

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synthesis or increased degradation, we first analyzed the time course of expression of the

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N-terminal product by Western blot (HA), which showed that levels of both full-length

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TK and the N-terminal product increased until about 10 hours post-infection (h.p.i.), and

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then plateaued, similar to ICP8 (Fig. 5A). We then performed a pulse-chase experiment

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by pulse-labeling infected cells with [35S]-methionine for 30 min at 5 h.p.i., followed by a

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chase in the presence of unlabeled methionine for 6 hours. HA-tagged products were then

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immunoprecipitated using anti-HA antibody for analysis. During the pulse phase, the N-

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terminal product from HA-G8-FLAG was labeled less efficiently than the full-length

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protein from HA-WT-FLAG. Using densitometric scanning and correcting for different

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methionine contents, the level of labeled N-terminal product was 13% of that of full-

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length protein, similar to the steady state level observed by Western blot. During the

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chase period, HA-WT-FLAG TK remained stable (Fig. 5B). There was some reduction

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in the amount of labeled N-terminal product from HA-G8-FLAG during the chase (Fig.

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5B). However, the reduction was only 2 fold over a 6 h period. This reduction is not

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sufficient to explain the 10-fold lower level of the N-terminal product, and explains why

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we saw no decrease of steady-state protein levels in the time course experiment (Fig. 5A).

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Thus, with a level of full-length TK of ~ 0.1% that of WT, and a level of N-

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terminal product of ~10% that of WT full-length TK, the frameshifting efficiency in G8

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mutant infected cells is about 1%, which is consistent with previous in vitro results using

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a dual-reporter assay (19).

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Analysis of nonsense mutations. To address where frameshifting occurs, we introduced

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nonsense mutations into sequences surrounding the G-string. Our reasoning was that if

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frameshifting occurs on the G-string, a stop codon immediately before it in the 0 reading

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frame (N-terminal frame) of the mutant, or immediately after it in the +1 frame of the

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mutant (the frame used after frameshifting) should block expression of full-length TK.

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We therefore constructed three mutant viruses: Stop 0a, with a stop codon in the 0

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reading frame immediately before the G-string, Stop +1b with a stop codon in the +1

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reading frame after the 7th G of the G-string, and Stop +1c, with a stop codon in the +1

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reading frame four codons further downstream (Fig. 6A and Fig. 7A, top). Each virus

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contained a FLAG-tag at the C-terminus of TK. Plaque autoradiogaphy showed no TK

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activity from any of these three nonsense mutants (data not shown). Lysates of cells

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infected with these viruses or with G8-FLAG contained similar amounts of N-terminal

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TK product, as assessed by Western blotting with anti-TK antiserum (Fig. 6B). The

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lysates were subjected to IP-Western (FLAG). In this case, we electrophoresed the IP'd

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proteins further into the SDS gel. This resolved two immunoreactive species in the G8-

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FLAG sample, a major species at the position of full-length TK, and a minor species with

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higher mobility (Fig. 6C). (We discuss the possible origin of the higher mobility species

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in the Discussion.) The Stop0a mutant expressed no detectable immunoreactive species

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(Fig. 6C). The Stop+1b and Stop+1c mutants expressed the minor, higher mobility

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species, but none migrating at the position of full-length TK, consistent with plaque

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autoradiography results (not shown). Thus, the behavior of these three mutants was

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consistent with our hypothesis that frameshifting occurs on the G-string (Fig. 7A, top),

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as mutations predicted to abolish full-length TK synthesis abolished expression of the

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polypeptide that co-migrated with full-length TK, and abolished TK activity.

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Using a similar approach, we also investigated the effects of a stop codon in the +1

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frame immediately upstream of the G-string (Stop +1a), and stop codons in the 0 frame

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after the sixth G in the G-string (Stop0b) or three codons downstream of the G-string

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(Stop0c). We reasoned that these mutations should not abolish frameshifting if that

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occurs on the G-string. Again, lysates of cells infected with these viruses expressed

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amounts of N-terminal TK product similar to that of G8-FLAG (Fig. 6B). The Stop+1a

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mutant, as predicted, exhibited TK activity by plaque autoradiography (data not shown),

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and expressed amounts of full-length TK polypeptide, and also the minor

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immunoreactive species, similar to those of G8-FLAG (Fig. 6C). The Stop0b and Stop 0c

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mutants did express full-length TK, in line with our hypothesis, although at reduced

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levels (Fig. 6C and Fig. 7A, bottom). Scanning densitometry relative to a dilution series

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indicate that full length TK expression was reduced ~5-fold. Interestingly, these mutants

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failed to express the higher mobility immunoreactive species (Fig. 6C). These data are

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consistent with frameshifting occurring on the G-string, with at least some frameshifting

336

occurring within the first 6G's of the G-string. However, they also indicate that

15

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sequences that are altered by the Stop0b and Stop0c mutations are important for

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frameshifting efficiency.

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16

340 341

Discussion To both evade drug therapy and cause disease, HSV ACVr mutants often use

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mechanisms to compensate for the effects of the mutations on TK activity. In this study,

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we examined the effects in HSV-infected cells on TK expression of three tk frameshift

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mutations found in clinical ACVr isolates using plaque autoradiography and a highly

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sensitive IP-Western technique. For one mutation, G9, our results confirm previous

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findings (12, 16) that reversion can account for much of the TK activity of these viruses

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(although it is likely that frameshifting also contributes to this (13, 16, 17), and extend

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those findings by detecting and quantifying full-length TK polypeptide synthesis from

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this mutant. For a second mutation, G6, we found that the mutation abolished detectable

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TK enzyme activity in a plaque autoradiography assay, and reduced full-length TK

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expression ~10,000-fold. For the third mutation, G8, we not only quantified its full-

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length TK expression, but explored the mechanisms that account for this expression.

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Using pulse-chase protocols, we determined that the ratio of synthesis of full-length TK

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to the N-terminal product resulting from the frameshift mutation -- i.e., the frameshifting

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efficiency -- was ~1%. By introducing nonsense mutations before and after the G-string,

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we found that frameshifting most likely occurs on the G-string. Of note, the G8 mutation

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reduced full-length TK expression substantially more than previous estimates (3, 15-18).

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We discuss our results with reference to mechanisms that compensate for tk frameshift

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mutations for TK expression and for latency and pathogenicity.

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The G6 mutation. To our knowledge, the TK activity of G6 mutants has not been

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previously assessed. We found that G6 exhibited no detectable TK activity in plaque

17

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autoradiography assays, and expressed ~10-fold less TK than the G8 mutant. In cultured

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cells, the G6 deletion mutation arises at roughly one-third the frequency of the G8 and G9

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insertion mutations together (C.B.C. Hwang, personal communication). However, in

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clinical isolates, the G6 mutation occurs roughly one tenth as frequently as G8 and G9

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mutants together (2, 5, 11, 27). We suggest that these differences in frequencies of

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isolation in cells vs. patients is because the G8 and G9 mutants express more TK, which

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can abet their replication and pathogenesis in vivo. We speculate that clinical G6 isolates

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compensate for their loss of TK by alleles in genes other than tk, as has been suggested

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for certain other clinical isolates (14, 18).

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Interestingly, the G6 motif is sufficient to mediate net +1 frameshifting in vitro

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(19), but it clearly mediates much less net -1 frameshifting in infected cells (this study)

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and very little if any in vitro (D.P. and D.M.C., unpublished results). In contrast, a

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mutant with a run of 7 G’s and a deletion of one G downstream (referred to as G7dG),

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which required -1 frameshifting for full-length TK expression, exhibited detectable TK

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activity (albeit lower than G8), and could reactivate in latently infected trigeminal ganglia

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with a low frequency (17). Our results thus suggest that the seventh G srongly enhances

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net -1 frameshifting on the G string.

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Mechanism of full-length TK expression in G8 mutant infected cells. The wild-type

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reading frame can be restored in cells infected with an insertion mutant such as G8 in

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three ways: DNA replication errors (reversion), transcription errors, and ribosomal

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frameshifting. The uniformity in the G8 population shown by both plaque

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autoradiography and IP-Western (FLAG) assays argues against a reversion mechanism

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for this mutant. We attempted to assess whether transcription errors could account for

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full-length TK expression by using reverse transcription-polymerase chain reaction

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followed by deep sequencing, and comparing the number of sequence reads containing

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G8 to those containing G7. However, we found that applying this approach to a synthetic

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RNA template containing G8 resulted in a frequency of G7 containing reads that was

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substantially greater than 0.1%, the level of full-length TK expressed by G8, making this

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assay difficult to interpret. Thus, we cannot exclude transcription errors as contributing

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to frameshifting in infected cells. However, in rabbit reticulocyte lysates, G7 containing

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transcripts occurred with a frequency of