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. 2016 Sep 29;90(20):9483-94.
doi: 10.1128/JVI.01547-16. Print 2016 Oct 15.

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Song Hee Lee  1 , Katie Caviness  2 , Emily R Albright  1 , Jeong-Hee Lee  1 , Christopher B Gelbmann  1 , Mike Rak  2 , Felicia Goodrum  2 , Robert F Kalejta  3
Affiliations

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

Song Hee Lee et al. J Virol. .

Abstract

The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34(+) hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency.

Importance: Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency but may do so in temporally distinct manners. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs.

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Figures

FIG 1
FIG 1
The UL138 gene is expressed as long and short isoforms. (A) Lysates from normal human dermal fibroblasts (HF) or THP-1 cells transfected with an empty vector (−) or with a plasmid expressing C-terminally HA-tagged UL138 (+) were harvested at 48 h posttransfection and analyzed by Western blotting with an HA antibody. GAPDH serves as a loading control. (B) Lysates from NHDFs mock infected (M) or infected with AD169-UL138HA at an MOI of 1 were collected at the indicated hours postinfection (hpi) and analyzed by Western blotting with an HA antibody. A long exposure is also shown. Tubulin served as a loading control. (C) VPA-treated THP-1 cells were mock infected (M) or infected with AD169-UL138HA (V) at an MOI of 3. At 24 h postinfection, lysates were collected and analyzed by Western blotting with an HA antibody. A long exposure is also shown. GAPDH served as a loading control.
FIG 2
FIG 2
pUL138-S initiates from methionine 16. (A) Amino acid sequence of full-length UL138. Potential in-frame methionine start sites are underlined. Putative transmembrane domain is shown in bold. (B) Schematic of UL138HA mutant alleles in the pSG5 expression vector. (C) Lysates from THP-1 cells transfected with an empty vector (EV) or the indicated UL138HA allele were collected at 48 h posttransfection and analyzed by Western blotting with an HA antibody. Tubulin served as a loading control. (D) Lysates from THP-1 cells transfected with an empty vector (EV) or plasmids encoding wild-type, Δ1–15, or M16I UL138HA were collected at 48 h posttransfection and analyzed by Western blotting with an HA antibody. Actin served as a loading control.
FIG 3
FIG 3
Ectopically expressed pUL138-L and pUL138-S localize to the Golgi apparatus. NHDFs (A) and THP-1 cells (B) were transfected with plasmids encoding wild-type (WT), Δ1–15, or M16I UL138HA virus. Twenty-four hours posttransfection, cells were stained with antibodies against HA and the Golgi body marker GM130 and imaged by indirect immunofluorescence. Nuclei were counterstained with Hoechst stain.
FIG 4
FIG 4
Both pUL138-L and pUL138-S repress an MIEP reporter in THP-1 cells. A luciferase reporter driven by the HCMV MIEP was cotransfected with a control TK-driven Renilla luciferase reporter and either an empty vector (EV) or an expression plasmid for wild-type, Δ1–15, or M16I UL138HA alleles in decreasing amounts from 3 μg to 0.25 μg. Total pSG5 transfected was brought up to 3 μg with pSG5 empty vector. The ratio of firefly to Renilla luciferase activity was determined 48 h posttransfection. Data represent means ± standard errors of the means (SEM) from three experiments. Lysates were analyzed by Western blotting with the indicated antibodies. Numbers under the 138HA Western blot represent 138HA band intensity when detectable relative to GAPDH intensity as quantified on LiCor Odyssey. Statistical comparisons were done between EV and UL138 isoforms with similar expression levels: ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant (P > 0.05) by Student's t test.
FIG 5
FIG 5
AD169-UL138HA mutant viruses express pUL138-L or pUL138-S. (A) Schematic of AD169-UL138HA wild-type, Δ1–15, and M16I viruses. (B) NHDFs were infected with the indicated virus at an MOI of 0.2, and cell-free virus was collected and analyzed by plaque assay at the indicated times. Data are the means ± SEM from 4 biological replicates. (C and D) Lysates from NHDFs (C) or MRC5s (D) mock infected or infected with the indicated virus at an MOI of 1 for 24 h were analyzed by Western blotting with an HA antibody. (E) Lysates from VPA-treated THP-1 cells mock infected or infected with the indicated virus at an MOI of 3 for 24 h were analyzed by Western blotting with an HA antibody. Long exposures are also shown. GAPDH served as a loading control.
FIG 6
FIG 6
UL138 proteins produced from AD169-based viruses localize to the Golgi apparatus. THP-1 (A) or VPA-treated primary CD34+ cells (B) were infected for 24 h with the indicated viruses at an MOI of 3 or 1, respectively, and then stained with antibodies for UL138 (HA) and the Golgi marker GM130 and imaged by indirect immunofluorescence. Nuclei were counterstained with Hoechst stain. (C) THP-1 cells treated with VPA were infected with AD-UL138HA-Δ1–15 at an MOI of 1 for 18 h and processed as described above.
FIG 7
FIG 7
UL138 isoforms suppress VPA-responsive IE transcription during experimental latency. (A) RNA from THP-1 cells infected with the indicated virus at an MOI of 1 in the absence (−) or presence (+) of 1 mM VPA for 18 h was analyzed by qRT-PCR for expression of IE exon 3. Viral gene expression was normalized to cellular GAPDH and expressed relative to untreated AD169-infected cells. Data are the means ± SEM from 4 biological replicates. Conventional RT-PCR analysis for the indicated genes is presented under the graph. (B) RNA from primary CD34+ cells infected with the indicated virus at an MOI of 1 in the absence (−) or presence (+) of 1 mM VPA for 24 h was analyzed by qRT-PCR for expression of IE exon 3 as described for panel A. Data are the means ± SEM from at least 4 biological replicates. Conventional RT-PCR analysis for the indicated genes is presented under the graph. *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.06) by two-sided Wilcoxon rank sum test.
FIG 8
FIG 8
TB40/E-UL138F mutant viruses express pUL138-L or pUL138-S. (A) Schematic of TB-UL138F wild-type, M1A, and M16A viruses. (B) MRC-5 fibroblasts were infected with the indicated virus at an MOI of 0.02, and virus accumulated in the cells and medium was quantitated by 50% tissue culture infective dose (TCID50) at the indicated times. Data are the means ± SEM from 3 biological replicates. (C) Lysates from MRC-5 fibroblasts mock infected or infected with the indicated virus at an MOI of 2 for 72 h were analyzed by Western blotting with the Flag antibody. Tubulin served as a loading control. (D) Lysates from THP-1 cells mock infected or infected with the indicated virus at an MOI of 4 for 72 h were analyzed by Western blotting with the Flag antibody. Tubulin served as a loading control.
FIG 9
FIG 9
UL138 proteins produced from TB40/E-based viruses localize to the Golgi apparatus and cytoplasm. (A) MRC5 fibroblasts were infected for 48 h with the indicated viruses at an MOI of 2 and stained with antibodies for UL138 (FLAG) and the Golgi marker GM130 and imaged by indirect immunofluorescence. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (B) THP-1 cells were infected for 48 h with the indicated viruses at an MOI of 2 and analyzed as described above.
FIG 10
FIG 10
pUL138-S suppresses progeny virion formation during HCMV latency in ESCs. (A) Infectious virions produced by ESCs infected with the indicated TB40/E-based viruses at an MOI of 2 for 10 days were quantified by plaque assay. Data from 5 biological replicates are graphed and shown in table format. *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.1) by two-sided Wilcoxon rank sum test. (B) Infectious virions produced by CD34+ cells infected with the indicated TB40/E-based viruses at an MOI of 2 for 10 days were quantified by TCID50 assay. Data from 2 biological replicates are graphed and shown in table format. Brackets represent the ranges of the data.
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References

    1. Boeckh M, Geballe AP. 2011. Cytomegalovirus: pathogen, paradigm, and puzzle. J Clin Invest 121:1673–1680. doi:10.1172/JCI45449. - DOI - PMC - PubMed
    1. Britt W. 2008. Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. Curr Top Microbiol Immunol 325:417–470. - PubMed
    1. Mocarski ES, Shenk T, Pass RF. 2007. Cytomegaloviruses, p 2701–2673. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed), Fields virology, 5th ed Lippincott, Williams & Wilkins, Philadelphia, PA.
    1. Goodrum F, Caviness K, Zagallo P. 2012. Human cytomegalovirus persistence. Cell Microbiol 14:644–655. doi:10.1111/j.1462-5822.2012.01774.x. - DOI - PMC - PubMed
    1. Sinzger C, Digel M, Jahn G. 2008. Cytomegalovirus cell tropism. Curr Top Microbiol Immunol 325:63–83. - PubMed

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