Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans.

BACKGROUND: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed. RESULTS: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag-Pro-Pol or Env were each present in multiple proviruses. CONCLUSIONS: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.     

Insertional polymorphisms of full-length endogenous retroviruses in humans

Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses in the human genome in that many HERV-K proviruses were inserted into the human germline after the human and chimpanzee lineages evolutionarily diverged [1, 2]. However, all full-length endogenous retroviruses described to date in humans are sufficiently old that all humans examined were homozygous for their presence [1]. Moreover, none are intact; all have lethal mutations [1, 3, 4]. Here, we describe the first endogenous retroviruses in humans for which both the full-length provirus and the preintegration site alleles are shown to be present in the human population today. One provirus, called HERV-K113, was present in about 30% of tested individuals, while a second, called HERV-K115, was found in about 15%. HERV-K113 has full-length open reading frames (ORFs) for all viral proteins and lacks any nonsynonymous substitutions in amino acid motifs that are well conserved among retroviruses. This is the first such endogenous retrovirus identified in humans. These findings indicate that HERV-K remained capable of reinfecting humans through very recent evolutionary times and that HERV-K113 is an excellent candidate for an endogenous retrovirus that is capable of reinfecting humans today.     

A Single Amino Acid Substitution in a Segment of the CA Protein within Gag That Has Similarity to Human Immunodeficiency Virus Type 1 Blocks Infectivity of a Human Endogenous Retrovirus K Provirus in the Human Genome

Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. However, no single HERV-K provirus in the human genome today appears to be infectious. Since the Gag protein is the central component for the production of retrovirus particles, we investigated the abilities of Gag from two HERV-K proviruses to support production of virus-like particles and viral infectivity. HERV-K113 has full-length open reading frames for all viral proteins, while HERV-K101 has a full-length gag open reading frame and is expressed in human male germ cell tumors. The Gag of HERV-K101 allowed production of viral particles and infectivity, although at lower levels than observed with a consensus sequence Gag. Thus, including HERV-K109, at least two HERV-K proviruses in human genome today have functional Gag proteins. In contrast, HERV-K113 Gag supported only very low levels of particle production, and no infectivity was detectable due to a single amino acid substitution (I516M) near the extreme C terminus of the CA protein within Gag. The sequence of this portion of HERV-K CA showed similarities to that of human immunodeficiency virus type 1 and other primate immunodeficiency viruses. The extreme C terminus of CA may be a general determinant of retrovirus particle production. In addition, precise mapping of the defects in HERV-K proviruses as was done here identifies the key polymorphisms that need to be analyzed to assess the possible existence of infectious HERV-K alleles within the human population.Approximately 8% of the human genome comprises endogenous retroviruses (ERVs) (33, 59). These viruses infect germ lineage cells and thereby enter the genome of the host species. Thus, endogenous proviruses (the integrated form of retroviral DNA) are transmitted from parents to offspring in genomic DNA. If ERV genomes are intact, viral particles may be generated that can reinfect the germ line and form proviruses at new positions in the host genome. However, ERVs are subject to the same mutagenic processes over evolutionary time as any cellular gene. In the absence of selective pressure on the host to maintain intact viral genomes, endogenous retroviral proviruses accrue mutations over evolutionary time that inactivate viral infectivity. Most of the ERVs in the human genome have converted to solo long terminal repeats (solo LTRs), which are the product of homologous recombination between LTRs at the ends of the complete viral genome. Other types of mutations, such as nucleotide substitutions, insertions, and deletions, can also affect ERV proviruses, and many of the retroviral proviruses in the human genome have been inactivated by such mutations, which created premature stop codons or frameshifts in viral open reading frames (ORFs). The vast majority of the ERVs present in humans today (and perhaps all of them) have incurred mutations that inactivated viral infectivity.One provirus that exists in the genome of approximately 20% of humans, human ERV K113 (HERV-K113, referred to here as K113), has full-length ORFs for all viral proteins (8, 63). However, this provirus does not appear to be infectious, as the pol and env genes of K113 do not support infectivity (9, 19, 20). K113 belongs to a subset of HERV-K called HML-2 (43). Since the human and chimpanzee lineages diverged about 6 million years ago (52), the only proviruses that entered the genome of the human lineage belong to this subgroup, although other members of this subgroup entered the germ line prior to the divergence of the human and chimpanzee lineages (8, 27, 44, 63). The human-specific proviruses of this subgroup are the most intact retroviruses in the human genome. Infectious HERV-K particles have been generated using two different approaches based on their DNA sequences. HERV-KCON (K-CON) was constructed based on the consensus sequence of human-specific HERV-K proviruses (34). Infectious HERV-K particles were also generated by combining pieces from three separate proviruses, HERV-K109 (K109) gag-pro, HERV-K115 pol, and HERV-K108 (K108) env (20). Thus, it may be that no single provirus is infectious, but recombination and/or genetic complementation among multiple genomic proviruses may be required to produce infectious HERV-K particles. This raises the questions of whether multiple functional HERV-K components exist in the human genome today and how close these components are to being able to form a functional viral genome that might be capable of reinfecting human cells.To begin addressing these issues, we examined two of the full-length HERV-K gag genes that exist in the human genome today. Like all retroviruses, HERV-K contains the four genes necessary for viral replication: gag, pro, pol, and env. The human-specific HERV-K proviruses exist in two forms, type I and type II (38, 39). The type II proviruses contain gag, pro, pol, and env plus an accessory gene, rec, that encodes a protein (Rec) that functions in nuclear export of unspliced viral RNA in a manner analogous to that of human immunodeficiency virus type 1 (HIV-1) Rev (12, 40, 41, 65, 66). In type I proviruses, the pol and env genes are fused in frame by a 292-bp deletion that includes the first coding exon of rec, and the viruses encode an additional protein called Np9 (5, 48). The gag genes are relevant to whether HERV-K components in the human genome today might form an infectious virus, as the Gag protein is sufficient to produce virus-like particles (VLPs) in the absence of other viral proteins (4). Formation of such particles is an essential step for subsequent viral replication. Therefore, we decided to investigate whether Gag proteins from K113 and a second provirus, HERV-K101 (K101), are active in functional assays.     

Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K.

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.     

HERV-K(OLD): ancestor sequences of the human endogenous retrovirus family HERV-K(HML-2)

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.