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Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K.

ABSTRACT: The human endogenous retrovirus family HTDV/HERV-K codes for the viral particles observed in teratocarcinoma cell lines. Two types of proviral genomes exist; these differ in the presence or absence of a stretch of 292 nucleotides. This sequence comprises the amino-terminal part of the env gene, the putative signal peptide, which overlaps in part with the carboxy terminus of the pol gene. Type 2 genomes containing this sequence presumably more closely reflect the structure of the infectious, replication-competent retrovirus ancestors of the HERV-K family than do type 1 genomes that lack the sequence. In human teratocarcinoma cell lines, both variants are expressed. Type 1 genomes, in which pol and env genes are fused, are deficient in splicing. Type 2 transcripts are spliced to subgenomic env mRNA and smaller messages. A doubly spliced transcript encodes a short open reading frame, preliminarily designated cORF (R. Löwer, K. Boller, B. Hasenmeier, C. Korbmacher, N. Mueller-Lantzsch, J. Löwer, and R. Kurth, Proc. Natl. Acad. Sci. USA 90:4480-4484). The genomic organization of cORF resembles that of nonprimate lentivirus rev genes: the first exon comprises nearly the entire signal peptide of env, and the second exon is derived from a different reading frame in the 3' part of the genome. A nucleolar localization signal, which is also a putative RNA binding domain, as well as a sequence with similarities to the Rev effector domain consensus sequence is present in the first exon. Secondary structure analysis reveals similarities to basic helix-loop-helix proteins. cORF is a small protein with an apparent molecular mass of 14 kDa which accumulates in the nucleolus as has been described for Rev proteins.

SUBMITTER: Lower R

PROVIDER: S-EPMC188557 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Project description:UnlabelledHuman endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied.ImportanceRetroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.

Project description:Human endogenous retroviruses (HERVs) are transcribed in many cancers including prostate cancer. Human endogenous retrovirus K (HERV-K) of the HML2 subtype is the most recently integrated and most intact retrovirus in the human genome, with many of the viral genomes encoding full- or partial-length viral proteins. To assess transcripts of HERV-K in prostate cancer cell lines and identify the specific HERV-K elements in the human genome that are transcribed, reverse transcriptase-PCR (RT-PCR) and cDNA sequencing were undertaken. Strand-specific RT-PCR, plasmid subcloning, and cDNA sequencing detected the presence of HERV-K(HML2) coding strand transcripts within four prostate cell lines (LNCaP, DU145, PC3, and VCaP). RT-PCR across splice junctions revealed splicing variants for env gene mRNA in three cell lines, two involving previously undescribed alternative splice sites. To determine the HERV-K loci from which the transcripts arose, RepeatMasker was used to compile a list of over 200 HERV-K internal genome segment fragments and over 1,000 HERV-K solo long terminal repeat (LTR) fragments in the human genome. Surprisingly, the sequences identified from internal positions of the viral genome were mostly smaller segments, while the LTRs were relatively intact. Possible reasons for this are discussed. The transcripts in the cell lines tested, arose from several HERV-K loci, with some proviruses being detected in multiple cell lines and others in only one of the four used. In some instances, transcripts from viral antisense strands was also detected. In addition, transcripts from both strands of solo LTRs were detected. These data show that transcripts from HERV-K loci commonly occur in prostate cancer cell lines and that transcription of either strand can occur. They also emphasize the importance of single nucleotide level analysis to identify the specific, individual HERV-K loci that are transcribed, and indicate that HERV-K expression in prostate cancer warrants further study.

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Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K.

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