Promoter expression of HERV-K (HML-2) provirus-derived sequences is related to LTR sequence variation and polymorphic transcription factor binding sites.

BACKGROUND: Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression. RESULTS: We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5' LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas. CONCLUSIONS: These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals.

Discovery of unfixed endogenous retrovirus insertions in diverse human populations.

Endogenous retroviruses (ERVs) have contributed to more than 8% of the human genome. The majority of these elements lack function due to accumulated mutations or internal recombination resulting in a solitary (solo) LTR, although members of one group of human ERVs (HERVs), HERV-K, were recently active with members that remain nearly intact, a subset of which is present as insertionally polymorphic loci that include approximately full-length (2-LTR) and solo-LTR alleles in addition to the unoccupied site. Several 2-LTR insertions have intact reading frames in some or all genes that are expressed as functional proteins. These properties reflect the activity of HERV-K and suggest the existence of additional unique loci within humans. We sought to determine the extent to which other polymorphic insertions are present in humans, using sequenced genomes from the 1000 Genomes Project and a subset of the Human Genome Diversity Project panel. We report analysis of a total of 36 nonreference polymorphic HERV-K proviruses, including 19 newly reported loci, with insertion frequencies ranging from <0.0005 to >0.75 that varied by population. Targeted screening of individual loci identified three new unfixed 2-LTR proviruses within our set, including an intact provirus present at Xq21.33 in some individuals, with the potential for retained infectivity.

The distribution of insertionally polymorphic endogenous retroviruses in breast cancer patients and cancer-free controls.

BACKGROUND: Integration of retroviral DNA into a germ cell can result in a provirus that is transmitted vertically to the host's offspring. In humans, such endogenous retroviruses (HERVs) comprise >8% of the genome. The HERV-K(HML-2) proviruses consist of ~90 elements related to mouse mammary tumor virus, which causes breast cancer in mice. A subset of HERV-K(HML-2) proviruses has some or all genes intact, and even encodes functional proteins, though a replication competent copy has yet to be observed. More than 10% of HML-2 proviruses are human-specific, having integrated subsequent to the Homo-Pan divergence, and, of these, 11 are currently known to be polymorphic in integration site with variable frequencies among individuals. Increased expression of the most recent HML-2 proviruses has been observed in tissues and cell lines from several types of cancer, including breast cancer, for which expression may provide a meaningful marker of the disease. RESULTS: In this study, we performed a case-control analysis to investigate the possible relationship between the genome-wide presence of individual polymorphic HML-2 proviruses with the occurrence of breast cancer. For this purpose, we screened 50 genomic DNA samples from individuals diagnosed with breast cancer or without history of the disease (n = 25 per group) utilizing a combination of locus-specific PCR screening, in silico analysis of HML-2 content within the reference human genome sequence, and high-resolution genomic hybridization in semi-dried agarose. By implementing this strategy, we were able to analyze the distribution of both annotated and previously undescribed polymorphic HML-2 proviruses within our sample set, and to assess their possible association with disease outcome. CONCLUSIONS: In a case-control analysis of 50 humans with regard to breast cancer diagnosis, we found no significant difference in the prevalence of proviruses between groups, suggesting common polymorphic HML-2 proviruses are not associated with breast cancer. Our findings indicate a higher level of putatively novel HML-2 sites within the population, providing support for additional recent insertion events, implying ongoing, yet rare, activities. These findings do not rule out either the possibility of involvement of such proviruses in a subset of breast cancers, or their possible utility as tissue-specific markers of disease.

Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses.

BACKGROUND: Integration of retroviral DNA into a germ cell may lead to a provirus that is transmitted vertically to that host's offspring as an endogenous retrovirus (ERV). In humans, ERVs (HERVs) comprise about 8% of the genome, the vast majority of which are truncated and/or highly mutated and no longer encode functional genes. The most recently active retroviruses that integrated into the human germ line are members of the Betaretrovirus-like HERV-K (HML-2) group, many of which contain intact open reading frames (ORFs) in some or all genes, sometimes encoding functional proteins that are expressed in various tissues. Interestingly, this expression is upregulated in many tumors ranging from breast and ovarian tissues to lymphomas and melanomas, as well as schizophrenia, rheumatoid arthritis, and other disorders. RESULTS: No study to date has characterized all HML-2 elements in the genome, an essential step towards determining a possible functional role of HML-2 expression in disease. We present here the most comprehensive and accurate catalog of all full-length and partial HML-2 proviruses, as well as solo LTR elements, within the published human genome to date. Furthermore, we provide evidence for preferential maintenance of proviruses and solo LTR elements on gene-rich chromosomes of the human genome and in proximity to gene regions. CONCLUSIONS: Our analysis has found and corrected several errors in the annotation of HML-2 elements in the human genome, including mislabeling of a newly identified group called HML-11. HML-elements have been implicated in a wide array of diseases, and characterization of these elements will play a fundamental role to understand the relationship between endogenous retrovirus expression and disease.

Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B.

Virus-induced membrane fusion can be subdivided into three phases defined by studies of class I and class II fusion proteins. During Phase I, two membranes are brought into close apposition. Phase II marks the mixing of the outer membrane leaflets leading to formation of a hemifusion intermediate. A fusion pore stably forms and expands in Phase III, thereby completing the fusion process. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins to complete membrane fusion, but none has been defined as class I or II. Therefore, we investigated whether HSV-1-induced membrane fusion occurred following the same general phases as those described for class I and II proteins. In this study we demonstrate that glycoprotein D (gD) and the glycoprotein H and glycoprotein L complex (gHL) mediated lipid mixing indicative of hemifusion. However, content mixing and full fusion required glycoprotein B (gB) to be present along with gD and gHL. Our results indicate that, like class I and II fusion proteins, fusion mediated by HSV-1 glycoproteins occurred through a hemifusion intermediate. In addition, both gB and gHL are probably directly involved in the fusion process. From this, we propose a sequential model for fusion via HSV-1 glycoproteins whereby gD is required for Phase I, gHL is required for Phase II, and gB is required for Phase III. We further propose that glycoprotein H and gB are likely to function sequentially to promote membrane fusion in other herpesviruses such as Epstein-Barr virus and human herpesvirus 8.

The nectin-1alpha transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins.

Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1alpha involved in cell fusion, we measured the ability of nectin-1alpha/nectin-2alpha chimeras, nectin-1alpha/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1alpha to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1alpha cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1alpha and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1alpha interaction in fusion.