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Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

ABSTRACT: The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.

SUBMITTER: Seifarth W

PROVIDER: S-EPMC189540 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

Seifarth W W Skladny H H Krieg-Schneider F F Reichert A A Hehlmann R R Leib-Mösch C C

Journal of virology 19951001 10

The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplifi ...[more]

PMID: 7545247

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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:BACKGROUND: Endogenous retroviral sequences (ERVs) are integral parts of most eukaryotic genomes and vastly outnumber exogenous retroviruses (XRVs). ERVs with a relatively complete structure were retrieved from the genetic archives of humans and chickens, diametrically opposite representatives of vertebrate retroviruses (over 3300 proviruses), and analyzed, using a bioinformatic program, RetroTector, developed by us. This rich source of proviral information, accumulated in a local database, and a collection of XRV sequences from the literature, allowed the reconstruction of a Pol based phylogenetic tree, more extensive than previously possible. The aim was to find traits useful for classification and evolutionary studies of retroviruses. Some of these traits have been used by others, but they are here tested in a wider context than before. RESULTS: In the ERV collection we found sequences similar to the XRV-based genera: alpha-, beta-, gamma-, epsilon- and spumaretroviruses. However, the occurrence of intermediates between them indicated an evolutionary continuum and suggested that taxonomic changes eventually will be necessary. No delta or lentivirus representatives were found among ERVs. Classification based on Pol similarity is congruent with a number of structural traits. Acquisition of dUTPase occurred three times in retroviral evolution. Loss of one or two NC zinc fingers appears to have occurred several times during evolution. Nucleotide biases have been described earlier for lenti-, delta- and betaretroviruses and were here confirmed in a larger context. CONCLUSION: Pol similarities and other structural traits contribute to a better understanding of retroviral phylogeny. "Global" genomic properties useful in phylogenies are i.) translational strategy, ii.) number of Gag NC zinc finger motifs, iii.) presence of Pro N-terminal dUTPase (dUTPasePro), iv.) presence of Pro C-terminal G-patch and v.) presence of a GPY/F motif in the Pol integrase (IN) C-terminal domain. "Local" retroviral genomic properties useful for delineation of lower level taxa are i.) host species range, ii.) nucleotide compositional bias and iii.) LTR lengths.

Project description:Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.

Dataset Information

Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

Publications

Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

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