Detection of T lymphocytes specific for human endogenous retrovirus K (HERV-K) in patients with seminoma

Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses that comprise about 8% of the human genome in that multiple HERV-K proviruses encode full-length viral proteins, and many HERV-K proviruses formed during recent human evolution. HERV-K gag proteins are found in the cytoplasm of primary tumor cells of patients with seminoma. We identified HERV-K-specific T cells in patients with a past history of seminoma using the interferon-gamma ELISPOT assay and an MHC-HERV-K peptide-specific tetramer. A minority of apparently healthy subjects without evident germ cell tumors also made HERV-K-specific T cell responses. In summary, we detected T cell reactivity to HERV-K peptides in both past seminoma patients and a minority of apparently healthy controls.     

The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.

Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.     

Detection of HERV-K(HML-2) viral RNA in plasma of HIV type 1-infected individuals

Approximately 8% of the human genome sequence is composed by human endogenous retroviruses (HERVs), most of which are defective. HERV-K(HML-2) is the youngest and most active family and has maintained some proviruses with intact open reading frames (ORFs) that code for viral proteins that may assemble into viral particles. Many HERV-K(HML-2) sequences are polymorphic in humans (present in some individuals but not in others) and probably many others may be unfixed (not inserted permanently in a specific chromosomal location of the human genome). In the present study HIV-1 and HCV-1-positive plasma samples were screened for the presence of HERV-K(HML-2) RNA in an RT-PCR using HERV-K pol specific primers. HERV-K(HML-2) viral RNA sequences were found almost universally in HIV-1(+) plasma samples (95.33%) but were rarely detected in HCV-1 patients (5.2%) or control subjects (7.69%). Other HERV-K(HML-2) viral segments of the RNA genome including gag, prt, and both env regions, surface (su), and transmembrane (tm) were amplified from HERV-K pol-positive plasma of HIV-1 patients. Type 1 and type 2 HERV-K(HML- 2) viral RNA genomes were found to coexist in the same plasma of HIV-1 patients. These results suggest the HERV-K(HML-2) viral particles are induced in HIV-1-infected individuals.     

A proposal for a new approach to a preventive vaccine against human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) is a more complex retrovirus, coding for several accessory proteins in addition to the structural proteins (Gag, Pol, and Env) that are found in all retroviruses. More complex retroviruses have not been isolated from birds, and simpler retroviruses have not been isolated from humans. However, the proviruses of many endogenous simpler retroviruses are present in the human genome. These observations suggest that humans can mount a successful protective response against simpler retroviruses, whereas birds cannot. Thus, humans might be able to mount a successful protective response to infection with a simpler HIV-1. As a model, a simpler bovine leukemia virus which is capable of replicating has been constructed; a simpler HIV-1 could be constructed in a similar fashion. I suggest that such a simpler HIV-1 would be a safe and effective vaccine against HIV-1.     

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of approximately 50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome approximately 30 million years ago.     

Human T-cell leukemia-lymphoma virus (HTLV) is in T but not B lymphocytes from a patient with cutaneous T-cell lymphoma.

A human type C retrovirus, designated HTLV, previously was isolated from or identified in some patients with leukemias and lymphomas of mature T lymphocytes. HTLV is genetically and serologically distinct from any known animal retroviruses. The absence of HTLV proviral sequences in DNA from normal humans showed that HTLV is not a ubiquitous endogenous (germ-line transmitted) virus of humans. Antibodies to HTLV core proteins have been identified in some people with T-cell neoplasias and are particularly prevalent in Japanese with adult T-cell leukemia, suggesting that HTLV is acquired horizontally. However, it was possible that HTLV is transmitted through the germ line of some (possibly rare) families and is then expressed in the HTLV- positive malignancies. An opportunity to study this question was provided by the development of several T-cell lines and a B-cell provided by the development of several T-cell lines and a B-cell line from one HTLV-positive patient with a cutaneous T-cell lymphoma. Here we report that HTLV proteins or nucleic acids (or both) are found in three independently derived T-cell lines, all shown by HLA typing to have originated from the patient. In contrast, the B-cell line, the identity of which was also ascertained by HLA typing, contained no detectable HTLV protein, RNA, or proviral DNA. Because the sensitivity of the latter assay is more than sufficient to detect one proviral equivalent per haploid genome, the results indicate that HTLV was not transmitted to this patient through the germ line but rather was acquired by infection.     

HERV-K113 is not associated with multiple sclerosis in a large family-based study

Numerous studies have invoked a role for retroviruses in multiple sclerosis (MS). Most have identified human endogenous retroviruses (HERVs) as possible etiological agents. The majority of HERVs originate from ancestral infection and then become progressively disabled by mutations over millions of years of primate evolution. Their presence in 100% of healthy humans, together with the paucity of functional retroviral genes, argues strongly against a causal role in disease. Recently, a new class of insertionally polymorphic HERVs has been described that is present in only a proportion of the population. One of them, HERV-K113, is notable for open reading frames for all of its genes and is found in 0-28% of humans with widespread geographic and racial variation. Thus HERV-K113 is a credible candidate for causing disease in a manner comparable to infectious retroviruses. Genomic DNA samples from 951 patients with MS were tested for the presence of the HERV-K113 allele by PCR, with their unaffected parents (n = 1902) acting as controls. HERV-K113 provirus was found in 70 out of 951 (7.36%) patients with MS and was not significantly increased compared to the combined parent group (6.52%). The results do not support an association between this endogenous retrovirus and MS. This study also emphasizes the need for large cohorts with controls for race and geographic location when examining possible links between polymorphic HERVs and disease.     

Isolation and phylogenetic analysis of HERV-K long terminal repeat cDNA in cancer cells.

Long terminal repeat (LTR) elements of human endogenous retrovirus (HERV-K) may have contributed to disease-associated structural change or genetic variation in the human genome. The LTR elements have been found to be coexpressed with sequences of closely located genes. We identified seven HERV-K LTR elements from mRNA of human cancer cells (HepG2, MCF7, and SiHa), using the RT-PCR approach. Four of them are closely related to the human-specific HERV-K LTR elements with a high degree of sequence homology in a neighbor-joining phylogenetic tree. The data suggest that recently proliferated HERV-K LTR elements are expressed actively in various cancer cells. These HERV-K LTR elements deserve further investigation as potential leads in the treatment of human cancer.     

A new endogenous primate type C virus isolated from the Old World monkey Colobus polykomos.

A new, genetically transmitted retrovirus has been isolated from the Old World monkey Colobus polykomos. This virus, designated CPC-1, is readily transmitted to both feline and human cells in culture. Nucleic acid hybridization studies reveal that there are 50-70 copies of the CPC-1 genome in colobus cellular DNA. Related virogene sequences can be detected in the DNA of all other Old World monkeys, as well as in the DNA of at least one ape species, the chimpanzee, indicating that this virus has been genetically transmitted in primates for 30-40 million years. CPC-1 is partially related to the type C virus previously isolated from stumptail monkeys (MAC-1). These two viruses have nucleic acid sequence homology, antigenic crossreactivity in their major viral structural protein, and a very similar host range in vitro. CPC-1 and MAC-1 therefore belong to the same class of genetically transmitted primate type C viruses and, as such, represent the first example in primates of analogous endogenous retroviruses isolated from two distantly related species.     

Long-term reinfection of the human genome by endogenous retroviruses

Endogenous retrovirus (ERV) families are derived from their exogenous counterparts by means of a process of germ-line infection and proliferation within the host genome. Several families in the human and mouse genomes now consist of many hundreds of elements and, although several candidates have been proposed, the mechanism behind this proliferation has remained uncertain. To investigate this mechanism, we reconstructed the ratio of nonsynonymous to synonymous changes and the acquisition of stop codons during the evolution of the human ERV family HERV-K(HML2). We show that all genes, including the env gene, which is necessary only for movement between cells, have been under continuous purifying selection. This finding strongly suggests that the proliferation of this family has been almost entirely due to germ-line reinfection, rather than retrotransposition in cis or complementation in trans, and that an infectious pool of endogenous retroviruses has persisted within the primate lineage throughout the past 30 million years. Because many elements within this pool would have been unfixed, it is possible that the HERV-K(HML2) family still contains infectious elements at present, despite their apparent absence in the human genome sequence. Analysis of the env gene of eight other HERV families indicated that reinfection is likely to be the most common mechanism by which endogenous retroviruses proliferate in their hosts.     

Insertional polymorphisms of endogenous HERV-K113 and HERV-K115 retroviruses in breast cancer patients and age-matched controls

Endogenous retroviral sequences resulting from ancient retrovirus infections of germline cells account for up to 8% of the human genome. Most of these sequences are highly truncated, have been altered by mutations, and do not encode functional genes. However, some members of the human endogenous retrovirus (HERV)-K family are remarkably intact and display high genetic homology to mouse mammary tumor virus (MMTV), a retrovirus causing breast cancer in mice. Two full-length HERVs (K113 and K115) have been reported to show insertional polymorphism. We used PCR to investigate the presence of these two HERVs in 102 female breast cancer patients and an equal number of age-matched controls with no history of malignancy (age range: 25-92 years). The two groups showed no significant difference in frequency (HERV-K113, 16.7% vs. 12.7%; HERV-K115, 4.9% vs. 9.8%) and no apparent association with histology, age at diagnosis, receptor status, HER-2/neu status, or TNM stage at diagnosis. This suggests that the two HERV-Ks do not play a pathogenetic role in the majority of breast cancer patients, though they may be involved in a minority of patients. The results are discussed.     

Phylogeny of a novel family of human endogenous retrovirus sequences, HERV-W, in humans and other primates

A novel human endogenous retrovirus, HERV-W, has been characterized on the basis of multiple sclerosis-associated retrovirus (MSRV) probes. We have analyzed the phylogenetic distribution of HERV-W in humans and other primate species. As HERV-W presents a C/D chimeric nature and is largely composed of deleted elements, Southern blots were performed using gag, pol, env, and LTR probes. The relative complexities observed for gag, pol, env, and LTR regions were similar in humans, apes, and Old World monkeys, the minimal number of bands observed after Southern blot analysis being 25, 50, 10, and at least 100, respectively. The HERV-W family entered the genome of catarrhines more than 25 million years ago.     

Construction of a clonable, infectious, and tumorigenic mouse mammary tumor virus provirus and a derivative genetic vector.

Molecular genetic studies of mouse mammary tumor virus (MMTV) have been hampered by the difficulty of cloning proviruses of milk-borne strains because of inhibitory sequences located in the gag gene. To surmount this barrier we have constructed a hybrid MMTV provirus composed of clonable 5' sequences (encompassing gag) from an endogenous MMTV provirus of C3H mice (Mtv-1) and 3' sequences from the milk-borne strain of MMTV in C3H mice, MMTV(C3H). Virions produced from XC cells transfected with this hybrid provirus are infectious in cell culture and tumorigenic in BALB/cJ mice. A vector derived from this provirus, containing the neomycin phosphotransferase gene (neo) and origins of replication from simian virus 40 and pBR322, is capable of transferring G418 resistance by virus infection in cell culture when supplied with viral proteins from either MMTV(C3H) or the hybrid MMTV. Expression of both hybrid and vector proviruses is inducible by dexamethasone in infected cells.     

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.     

Human T-cell leukemia-lymphoma virus (HTLV): cloning of an integrated defective provirus and flanking cellular sequences.

Human T-cell leukemia-lymphoma virus (HTLV) is the first unequivocal human retrovirus. Seroepidemiological and virus isolation studies indicate that HTLV is etiologically associated with a subtype of adult T-cell malignancy. We have molecularly cloned approximately 1 kilobase of sequences derived from the 5' and 3' termini of the HTLV genome. Use of these clones as probes allowed isolation of a 9.8-kilobase EcoRI fragment from a genomic DNA library of an HTLV-infected neoplastic T-cell line (CR). Analysis of this clone revealed the presence of cellular sequences flanking approximately 5 kilobases of viral sequences including one long terminal repeat sequence. The 5' and 3' clones, as well as subclones derived from different regions of the genomic clone, were used as probes to compare integrated proviruses and viral RNA expression in different HLTV-infected neoplastic T cell lines. The results indicate that the infected cells are of clonal origin with respect to the virus integration sites and they express multiple viral mRNA species including a 35S RNA.