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. 2004 Jun;78(12):6585-94.
doi: 10.1128/JVI.78.12.6585-6594.2004.

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

Ke Lan  1 , Daniel A Kuppers, Subhash C Verma, Erle S Robertson
Affiliations

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

Ke Lan et al. J Virol. 2004 Jun.

Abstract

Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

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Figures

FIG. 1.
FIG. 1.
Scheme showing structurally important domains of LANA, Rta, and the Rta promoter. (A) As shown, LANA is a 1,162-amino-acid protein (strain BC-1). Numbers indicate amino acids (aa). Putative domains include the following: an N-terminal proline-rich domain (P-rich); an aspartic acid, glutamic acid repeat region (DE); glutamine-, glutamic acid-, and proline-rich repeats (QEP); glutamine-, arginine-, glutamic acid-, and proline-rich repeats (QFRP); glutamine-, aspartic acid-, and glutamic acid-rich repeats (QDE); and a leucine zipper (LZ). LANA also appears to have NLSs in both the amino- and carboxy-terminal regions, and a DNA binding domain (DBD) has been mapped to the distal carboxy terminus (13, 45). (B) The various known functional domains of the Rta protein. Three domains—the DNA binding and dimerization domain, located at the amino terminus (530 aa), the proline-rich domain, located in the middle, and the acidic activation domain, located in the carboxy terminus—are shown. Two NLSs are indicated: one at the amino terminus and one located at aa 514 to 528 of the Rta protein. AD1 through AD4 represent the conserved motifs that are essential for the Rta transactivation function (57). (C) Scheme showing the Rta promoter used in the present study. Putative transcriptional factor binding sites are shown. Numbers indicate nucleotides according to the KSHV genome of BC-1 (15, 25, 26, 45).
FIG. 2.
FIG. 2.
LANA represses the transcriptional activity of the Rta gene promoter in human cells. The pRpluc reporter plasmid contains a 3-kb sequence upstream of the translation initiation site of the rta gene that drives the expression of firefly luciferase (15). Five micrograms of the pRpluc reporter plasmid was cotransfected into HEK 293 cells (A) or Burkitt lymphoma BJAB cells (B) with either 2.5, 5, 10, or 15 μg of pA3M-LANA. Total transfected DNA was normalized with the pA3 M vector. Promoter activity was expressed as the fold activation relative to activity with pRpluc alone (control). Means and standard deviations from three independent transfections are shown. Protein lysates were analyzed by Western blotting for levels of expression of transfected protein with the Myc monoclonal antibody to detect LANA and the RTA monoclonal antibody to detect Rta protein. Protein levels were shown by Ponceau S staining.
FIG. 3.
FIG. 3.
LANA represses Rta-mediated autoactivation. Ten million HEK 293 cells (A) or Burkitt lymphoma BJAB cells (B) were transfected with 5 μg of the pRpluc luciferase reporter construct, 10 μg of pCR3.1-Rta, and 2.5, 5, 10, or 15 μg of the pA3M-LANA expression construct as indicated. Total transfected DNA was normalized with the pA3M-vector. Promoter activity was expressed as the fold activation relative to activity with pRpluc alone (control). Means and standard deviations from three independent transfections are shown. Protein lysates were analyzed by Western blotting for levels of expression of transfected protein with the Myc monoclonal antibody to detect LANA and the Rta monoclonal antibody to detect Rta protein. The protein lysate was used as a loading control and was stained with Ponceau S.
FIG. 4.
FIG. 4.
LANA down-regulates KSHV replication and virion production through repression of Rta. Ten million BC3 cells were transfected with 2.5, 5, or 10 μg of the LANA-Myc expression vector. At 12 h posttransfection, cells were induced with TPA and butyrate. At 48 h (A) or 72 h (B) postinduction, supernatants of transfected cells were harvested for PCR to check the virion production level of KSHV. The relative densities of the bands from the PCR products were measured with ImageQuant software (Molecular Dynamics). Cell lysates were analyzed by Western blotting (WB) for levels of expression of transfected protein with the Myc monoclonal antibody to detect LANA and the Rta monoclonal antibody to detect Rta protein. The protein lysate was used as a loading control and was stained with Ponceau S. Lanes 1, mock transfection; lanes 2, 10.0 μg of the pA3M-vector; lanes 3, 2.5 μg of pA3M-LANA and 7.5 μg of the pA3M-vector; lanes 4, 5.0 μg of pA3M-LANA and 5.0 μg of the pA3M-vector; lanes 5, 10.0 μg of pA3M-LANA.
FIG. 5.
FIG. 5.
Immunoprecipitation analysis with a mouse anti-Myc antibody or a mouse anti-Rta antibody showed that Rta was directly immunoprecipitated with LANA in BJAB (A) and 293T (B) cells. For each experiment, 30 million cells were either cotransfected with Rta and LANA-Myc expression vectors or were transfected with one of these vectors. At 24 h posttransfection, cells were harvested and lysed; lysates were used for immunoprecipitation analysis. L, lysate; PC, precleared lysate; IP, immunoprecipitate; WB, Western blotting..
FIG. 6.
FIG. 6.
LANA interacts with endogenously induced Rta in KSHV-infected pleural effusion lymphoma cells. Immunoprecipitation analysis with a polyclonal rabbit antiserum against Rta showed that LANA was directly immunoprecipitated with Rta in BC3 (A) and BCBL1 (B) cells. For each experiment, 100 million cells were induced with 20 ng of TPA/ml and 1.5 mM sodium butyrate. At 48 h postinduction, cell lysates were used for immunoprecipitation analysis. L, lysate; PC, precleared lysate; IP, immunoprecipitate; WB, Western blotting.
FIG. 7.
FIG. 7.
The carboxy-terminal region of LANA interacts with Rta in vitro. Full-length LANA and Rta as well as LANA clones with truncations in the carboxy- and amino-terminal regions were in vitro transcribed and translated. 35S-labeled products were incubated with GST as well as either GST-LANA or GST-Rta. Pulldown products were electrophoresed on 8% SDS-PAGE gels, dried, and exposed to a PhosphorImager. Input controls of 10% for LANA and 5% for Rta were run as well. A schematic for the LANA clones used is shown below the gels. AD, acidic domain; LZ, leucine zipper; DBD, DNA binding domain.
FIG. 8.
FIG. 8.
Immunofluorescence analysis showed that Rta was localized to the same nuclear compartment as LANA in different cells. (A) BJAB or 293T cells were cotransfected with 10 μg each of the pCR3.1-Rta and pA3 M-LANA expression vectors. At 24 h posttransfection, cells were harvested for immunofluorescence analysis. (B and C) Uninduced (Un) or induced (In) BC3 and BCBL1 cells were also used for immunofluorescence analysis. For induction, BC3 and BCBL1 cells were treated with 10 ng of TPA/ml and 1.5 mM butyrate. After 24 or 48 h of treatment, cells were harvested. LANA is expressed in almost every cell, and Rta is expressed in some induced cells.
FIG. 9.
FIG. 9.
Hypothetical model for LANA inhibition of lytic replication by targeting of Rta. Rp, Rta promoter; M, methylation.
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