J Bacteriol Virol.  2006 Dec;36(4):237-245. 10.4167/jbv.2006.36.4.237.

Expression and Packaging of a Human Endogenous Retrovirus-K Genomic DNA Clone

• Ha-Lee YM

• KMID: 2055032
• DOI: http://doi.org/10.4167/jbv.2006.36.4.237

Abstract

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.

Keyword

HERV-K; Genome; Gag; Expression; Packaging

MeSH Terms

Autoimmune Diseases
Capsid Proteins
Cell Line
Clinical Coding
Clone Cells*
DNA*
Endogenous Retroviruses
Genome
Genome, Human
Humans*
Nuclear Export Signals
Product Packaging*
Proviruses
Retroviridae
RNA
Transfection
Capsid Proteins
DNA
Nuclear Export Signals
RNA

Figure

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  Figure 1. Cloning and expression of full-length HERV-K108. (A) The schematic map of HERV-K108. (B) The 293T, COS-1 and D17 cells were transected with no DNA, the full-length HERV-K 108 genomic clone (pF9 and pF12), or replication competent MPMV clone (MPMV). 2 days after transfection, the VLPs were collected by centrifugation and the presence of gag proteins was analyzed by western blot analysis by anti-HERV gag antibody. The processed MPMV gag proteins, p27, p17, could also be detected by anti-HERV gag antibody due to cross-reactivity.

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  Figure 2. Gag expression requires nuclear export signal. (A) the schematic drawing of gag expression clones. The small triangles indicate the location of start and stop codons. His, histidine tag; HA, hemagglutinin tag; CTE, constitutive transport element. (B and C) 293T cells were transfected with various constructs including gag expression clones. 2 days after transfection the supernatants were collected and analyzed by western analysis with anti-HERV gag antibody (B). 1. no DNA; 2. pSV-lac (negative plasmid control); 3. pKgag13A-HA; 4. pKgag13AHA-CTE. (C) Western analysis with anti-MPMV gag antibody. 1. no DNA; 2. pMPgag3A-His; 3. pMPgag13A-HA; 4. pMPgag13A-CTE. The arrows on the right indicate gag proteins.

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  Figure 3. Construction and expression of LacZ reporter constructs. (A) the schematic drawing of LacZ expression reporter. The triangles indicate the location of translation stop site. (B) LacZ reporters were transfected into 293T and Tera-1 cells and the LacZ expression was analyzed in-situ X-gal staining as described in Materials and Methods.

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  Figure 4. co-transfection of LacZ reporter with gag expression clones. (A) 293T cells were transfected either with (1) pK-lac or (2) pK-lac and pKgag13A-CTE. Two days later, the supernatant were assayed for the presence of gag VLP by western analysis using anti-HERV gag antibody, (B) 293T cells were transfected either with (1) pMP-lac, or (2) pMP-lac and pMPgag3A-CTE and analyzed with anti-MPMV gag antibody. The arrows on the left indicate gag proteins.

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  Figure 5. Packaging of reporter RNA by gag VLP. (A) Total reporter RNA in 293T cells. 293T cells were transfected with (1). pMP-lac, (2) pMP-lac and pMPgag3A-CTE, (3) pMP-lac and pMPgag3A-CTE, (4) pK-lac, (5) pK-lac and pKgag13A-CTE. 2 days after transfection, total cellular RNA was prepared and analyzed by RT-PCR with a LacZ primer set (B) MPMV reporter RNA in MPMV gag VLP. 293T cells were transfected with (1) pMP-lac, (2) pMP-lac and pMPgag3A-CTE, (3) pMP-lac and pMPgag3A-CTE, (4) pK-lac, (5) pK-lac and pKgag13A-CTE. Two days after transfection, supernatant VLP was prepared and analyzed by RT-PCR. First amplification was carried out using a MPMV specific primer set and second amplification was with lacZ nested primer set. (C) HERV-K reporter RNA in HERV gag VLP. 293T cells were transfected with (1) pK-lac and pKgag13A, (2) pMP-lac and pMPgag3A-CTE, (3) pK-lac and pKgag13A-HA, (4) pK-lac and pKgag13A-CTE. 2 days after transfection, supernatant VLP was analyzed by RT-PCR. First amplification was carried out using an HERV-K108 specific primer set and the second with LacZ nested primer set; C, control DNA (pK-lac).

Reference

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