The human genome represents a fossil record of ancient retroviruses that once replicated in the ancestors of contemporary humans. Indeed, approximately 8% of human DNA is composed of sequences that are recognizably retroviral. Despite occasional reports associating human endogenous retrovirus (HERV) expression with human disease, almost all HERV genomes contain obviously inactivating mutations, and none are thought to be capable of replication. Nonetheless, one family of HERVs, namely HERV-K(HML-2), may have replicated in human ancestors less than 1 million years ago. By deriving a consensus sequence, we reconstructed a proviral clone (HERV-KCON) that likely resembles the progenitor of HERV-K(HML-2) variants that entered the human genome within the last few million years. We show that HERV-KCON Gag and protease proteins mediate efficient assembly and processing into retrovirus-like particles. Moreover, reporter genes inserted into the HERV-KCON genome and packaged into HERV-K particles are capable of infectious transfer and stable integration in a manner that requires reverse transcription. Additionally, we show that HERV-KCON Env is capable of pseudotyping HIV-1 particles and mediating entry into human and nonhuman cell lines. Furthermore, we show that HERV-KCON is resistant to inhibition by the human retrovirus restriction factors tripartite motif 5α and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 3G but is inhibited by APOBEC 3F. Overall, the resurrection of this extinct infectious agent in a functional form from molecular fossils should enable studies of the molecular virology and pathogenic potential of this ancient human retrovirus.

At least 8% of the human genome was formed by integration of retroviral DNA sequences. Here we analyze the forces directing the accumulation of human endogenous retroviruses (HERVs) by comparing de novo HERV integration targeting with the distribution of fixed HERV elements in the human genome. All known genomic HERVs are inactive due to mutation, but we were able to study integration targeting using a reconstituted consensus HERV-K (designated HERV-K Con ). We found that HERV-K Con integrated preferentially in transcription units, in gene-rich regions, and near features associated with active transcription units and associated regulatory regions. In contrast, genomic HERV-K proviruses are found preferentially outside transcription units. The minority of genomic HERVKs present inside transcription units are in opposite transcriptional orientation relative to the host gene, the orientation predicted to be minimally disruptive to host mRNA synthesis, but de novo HERV-K Con integration within transcription units showed no orientation bias. We also found that the youngest HERV-K elements in the human genome showed a distribution intermediate between de novo HERV-K Con integration sites and older fixed HERV-Ks. These findings indicate that accumulation of HERVs in the human germline is a twostep process: integration targeting biases direct initial accumulation, then purifying selection leads to loss of proviruses disrupting gene function.[Keywords: HERV-K; HML2; genome construction; integration; positive selection] Supplemental material is available at http://www.genesdev.org.

Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome, but all are remnants of ancient retroviral infections and harbor inactivating mutations that render them replication defective. Nevertheless, as viral "fossils," HERVs may provide insights into ancient retrovirus-host interactions and their evolution. Indeed, one endogenous retrovirus [HERV-K(HML-2)], which has replicated in humans for the past few million years but is now thought to be extinct, was recently reconstituted in a functional form, and infection assays based on it have been established. Here, we show that several human APOBEC3 proteins are intrinsically capable of mutating and inhibiting infection by HERV-K(HML-2) in cell culture. We also present striking evidence that two HERV-K(HML-2) proviruses that are fixed in the modern human genome (HERV-K60 and HERV-KI) were subjected to hypermutation by a cytidine deaminase. Inspection of the spectrum of mutations that are found in HERV-K proviruses in the human genome and HERV-K DNA generated during in vitro replication in the presence of each of the human APOBEC3 proteins unequivocally identifies APOBEC3G as the cytidine deaminase responsible for hypermutation of HERV-K60 and HERV-KI. This is a rare example of the antiretroviral effects of APOBEC3G in the setting of natural human infection, whose consequences have been fossilized in human DNA, and a striking example of inactivation of ancient retroviruses in humans through enzymatic cytidine deamination.