Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12

Porter, Iain M.; Stow, Nigel D.

doi:10.1099/vir.0.80403-0

Cited by 11 publications

(10 citation statements)

References 40 publications

(48 reference statements)

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“…The phenotype of UL12 mutants is complex. We previously reported that the UL12-null virus, AN-1, was compromised for growth on nonpermissive cells, with viral yields 2 to 3 logs lower than that of wild-type virus (33)(34)(35)55). In this paper, we show that the growth defect of AN-1 in nonpermissive cells is even more serious than previously recognized.…”

Section: Discussionmentioning

confidence: 52%

The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus

Grady

Szczepaniak

Murelli

et al. 2017

J Virol

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The Herpes simplex virus type I alkaline nuclease, UL12, has 5' to 3' exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12 null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12-D340E (D340E), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA containing capsids and more empty capsids than wild type virus. In addition, DNA packaged by the viral mutants was aberrant as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic α-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered as a target for development of antiviral agents. Herpes Simplex Virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV1/2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.

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Section: Discussionmentioning

confidence: 52%

The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus

Grady

Szczepaniak

Murelli

et al. 2017

J Virol

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“…UL12 is another HSV-1 protein with a nuclease motif and for which nuclease activity has been confirmed. UL12 may play a role in resolving X and Y DNA structures arising during viral DNA replication and in encapsidation (32). Nuclease activity has not yet been reported for UL24; however, the discovery that the motif is important for the function of the protein suggests that UL24 may indeed possess a nuclease function.…”

Section: Discussionmentioning

confidence: 99%

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

Bertrand

Leiva‐Torres

Hyjazie

et al. 2010

J Virol

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The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2,7,34,36,39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11,13,15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/ E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm a...

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“…The growth of baby hamster kidney 21 clone 13 (BHK) cells and preparation of stocks of wild-type (wt) HSV-1, strain 17 syn + , were as described previously (Porter & Stow, 2004). Rabbit skin cells were grown in Dulbecco's modified Eagle's medium containing 10 % fetal calf serum, 100 U penicillin ml −1 and 100 μg streptomycin ml −1 .…”

Section: Methodsmentioning

confidence: 99%

The UL15 protein of herpes simplex virus type 1 is necessary for the localization of the UL28 and UL33 proteins to viral DNA replication centres

Higgs

Preston

Stow

2008

Journal of General Virology

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The UL15, UL28 and UL33 proteins of herpes simplex virus type 1 (HSV-1) are thought to comprise a terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. Immunofluorescence studies confirmed that shortly after infection with wild-type HSV-1 these three proteins localize to viral DNA replication compartments within the nucleus, identified by the presence of the single-stranded DNA-binding protein, ICP8. In cells infected with either UL28- or UL33-null mutants, the other two terminase proteins also co-localized with ICP8. In contrast, neither UL28 nor UL33 was detectable in replication compartments following infection with a UL15-null mutant, although Western blot analysis showed they were present in normal amounts in the infected cells. Provision of UL15 in a complementing cell line restored the ability of all three proteins to localize to replication compartments. These data indicate that UL15 plays a key role in localizing the terminase complex to DNA replication compartments, and that it can interact independently with UL28 and UL33.

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Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12

Cited by 11 publications

References 40 publications

The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus

The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

The UL15 protein of herpes simplex virus type 1 is necessary for the localization of the UL28 and UL33 proteins to viral DNA replication centres

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