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. 2000 Jan;74(2):934-43.
doi: 10.1128/jvi.74.2.934-943.2000.

Potential role for luman, the cellular homologue of herpes simplex virus VP16 (alpha gene trans-inducing factor), in herpesvirus latency

R Lu  1 , V Misra
Affiliations

Potential role for luman, the cellular homologue of herpes simplex virus VP16 (alpha gene trans-inducing factor), in herpesvirus latency

R Lu et al. J Virol. 2000 Jan.

Abstract

The cascade of herpes simplex virus (HSV) gene expression that results in viral replication begins with the activation of viral immediate-early (IE) genes by the virion-associated protein VP16. VP16 on its own is inefficient at associating with complexes formed on IE gene promoters and depends upon the cellular factor HCF for its activity. In this respect VP16 mimics the host basic leucine zipper (bZIP) protein Luman, which also requires HCF for activating transcription. Our objective is to explore interactions between Luman and HCF and to determine if they play a role in the biology of herpesviruses. In this report we show that in cultured cells ectopically expressed Luman was retained in the cytoplasm, where it colocalized with Calnexin, a protein normally associated with the endoplasmic reticulum (ER). Retention of Luman in the ER depends on a hydrophobic segment of the protein that probably serves as a transmembrane domain. Deletion of this domain changed the intracellular location of Luman so that most of the mutant protein was in the nucleus of cells. While HCF was present in the nucleus of most cells, in cells expressing Luman it was retained in the cytoplasm where the two proteins colocalized. This cytoplasmic association of Luman and HCF could also be demonstrated in neurons in trigeminal ganglia removed from cattle soon after death. Cells in tissue culture that expressed Luman, but not a mutant form of the protein that fails to bind HCF, were resistant to a productive infection with HSV type 1 (HSV-1). We hypothesize that similar Luman-HCF interactions in sensory neurons in trigeminal ganglia result in the suppression of viral replication and the establishment of latency. Interestingly, Luman could activate the promoters of IE110 and LAT, two genes that are critical for reactivation of HSV-1 from latency. This suggests a role for Luman in the reactivation process as well.

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Figures

FIG. 1
FIG. 1
Location of Luman and Calnexin in transfected cells. HeLa cells were transfected with either pcLuman (A and B) or pcLumanΔTm (C and D). Cells were stained 48 h later with a mixture of anti-FLAG monoclonal antibodies which recognize FLAG-tagged Luman and rabbit anti-Calnexin antibodies, followed by a mixture of Alexa488-tagged anti-rabbit and Alexa546-tagged anti-mouse antibodies. The cells were visualized in a fluorescent microscope by using either a 546-nm (A and C) or a 450- to 490-nm (B and D) filter.
FIG. 2
FIG. 2
Putative transmembrane domain of Luman. The top figure shows the results of TmPRED analysis of the amino acid sequence of Luman. Portions of the protein with positive values (above the dotted line) have the potential of forming transmembrane domains. The portion of the Luman protein, residues 229 to 245, that display a high probability of forming a Tm domain are shown. The bottom figure is a schematic diagram of Luman and the ΔTm and 1–220 mutants showing their functional domains. Domains: a, activation; h, HCF binding; l, DNA binding; l, dimerization (leucine zipper); t, putative transmembrane.
FIG. 3
FIG. 3
Location of Luman and HCF in transfected cells. HeLa cells were transfected with either pcLuman (A and B) or pcLuman(Y81A) (C and D). Cells were stained 48 h later with a mixture of anti-FLAG monoclonal antibodies which recognize FLAG-tagged Luman and rabbit anti-HCF, followed by a mixture of Alexa488-tagged anti-rabbit and Alexa546-tagged anti-mouse antibodies. The cells were visualized in a fluorescent microscope by using either a 546-nm (A and C) or a 450- to 490-nm (B and D) filter.
FIG. 4
FIG. 4
Location of Luman and HCF in trigeminal ganglia. Trigeminal ganglia were removed from a cow within 30 min of death, embedded, and frozen in liquid nitrogen. Consecutive 6-μm sections were either stained with hematoxylin-eosin (A) or for Luman (B) or HCF (C). Rabbit anti-Luman antibodies and Alexa546-tagged anti-rabbit antibodies were used to visualize Luman, while rabbit anti-HCF and Alexa488-tagged anti-rabbit antibodies were used to visualize HCF. Corresponding fields from the three sections were located and photographed by using the appropriate filters.
FIG. 5
FIG. 5
Luman protects cells from HSV in an HCF-dependent manner. HeLa cells were transfected with either pcLuman (A and B) or pcLuman(Y81A) (C and D). At 48 h after transfection cells were infected with HSV-1 (KOS) at a multiplicity of about 10 PFU/cell. Then, 20 h later, cells were fixed and stained with a mixture of anti-HSVgC monoclonal antibodies and rabbit anti-Luman antibodies, followed by a mixture of Alexa488-tagged anti-mouse and Alexa546-tagged anti-rabbit antibodies. The cells were visualized in a fluorescent microscope by using either a 546-nm (A and C) or a 450- to 490-nm (B and D) filter.
FIG. 6
FIG. 6
Luman activates the HSV IE110 promoter in an HCF-dependent and octamer-GARAT-independent manner. HeLa cells were transfected with target plasmid pAB2, which has the minimal IE110 promoter linked to coding sequences for CAT, or pAB2-167, which in addition has an IE110 octamer-GARAT motif. The cells were also transfected with pcDNA3-0 or pcDNA3 expressing Luman-Lu, Luman(Y81A)-Lu(Y81A), or VP16-VP16. At 48 h after transfection cells were lysed and assayed for CAT.
FIG. 7
FIG. 7
Activation of the HSV IE110 promoter requires the putative CRE element. HeLa cells were transfected with target plasmid pAB2, which has the minimal IE110 promoter linked to coding sequences for CAT, or pAB2 in which two nucleotides of the CRE site have been altered (indicated). The cells were also transfected with pcLuman. At 48 h after transfection cells were lysed and assayed for CAT.
FIG. 8
FIG. 8
Luman activates the HSV LAT promoter. HeLa cells were transfected with the target plasmid pBB15, which contains 138 bp upstream from the start of transcription of LAT and either pcDNA-0 or pcLuman-Lu. At 48 h after transfection cells were lysed and assayed for CAT.
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