Wednesday, February 21, 2007
An ancient retrovirus is resurrected
Retroviruses have been around longer than humanity itself. In fact, the best-known family member, HIV, is a relative youngster, with its first known human infections occurring sometime in the mid-20th century. But although many retroviruses went extinct hundreds of thousands or millions of years ago, researchers studying the pathogens don't use the traditional tools of paleontologists: They need look only as far as our own DNA. Retroviruses infect cells and replicate by inserting their DNA into their host cell's genome. If that cell happens to be a germ cell, such as a sperm, an egg or their precursors, then the retroviral DNA is inherited by offspring just like a normal gene. Humans have many defunct retroviruses deposited in our DNA, remnants of ancient retroviruses that replicated in our ancestors millions of years ago. Now, researchers have brought one of those retroviruses back to life.
"In our DNA, there's a fossil record of retroviruses that used to infect us," says Paul Bieniasz, associate professor and head of the Laboratory of Retrovirology at Rockefeller University and the Aaron Diamond AIDS Research Center [Aids]. In fact, about eight percent of human DNA is made up of retroviral sequences. Bieniasz and Youngnam Lee, a graduate student in the Bieniasz lab, have excavated some of that DNA and - in an attempt to better understand how humans and retroviruses co-evolved - they have resurrected an ancient retrovirus, one that can create new viral particles and infect human cells. They describe their work in a paper published by PLoS Pathogens last month .
The extinct retroviruses embedded in our DNA can't reproduce because of mutations in one or more of their genes. The younger of these human endogenous retroviruses (or HERVs) have fewer changes, and judging by the paucity of genetic alterations, at least one subfamily - HERV-K - was likely still active less than a few hundred thousand years ago. Different members of this subfamily have slightly different mutations. "But as of a few months ago," Bieniasz says, "there was no replication-competent form of this virus."
To eliminate those mutations that kept HERV-K from replicating, the two researchers deduced a genetic sequence that was a consensus of 10 different HERV-K proviruses (provirus) and synthesized the whole viral genome from scratch. Then, they took that sequence (which they dubbed HERV-KCON) and inserted it into cultured human cells to see if it would result in the creation of HERV-K structural proteins. Their consensus sequence resulted in not only functional proteins, but in a retrovirus that was capable of creating new viral particles and integrating itself into a host cell's genome. "This is the first time this has been done with a viral genome that was effectively dead, and now is alive - or at least has all the functions that suggest it should replicate," Bieniasz says.
The project began, Lee says, because certain human and non-human primate cells produce proteins that appear to block HIV from replicating. "And the question is where did the proteins come from?" she asks. "By studying these extremely old viruses, we can tap into what happened in our ancestors millions and millions of years ago."
Source: Rockefeller University PR February 20, 2007
 Reconstitution of an Infectious Human Endogenous Retrovirus
Young Nam Lee and Paul D. Bieniasz
Citation: Lee YN, Bieniasz PD (2007) Reconstitution of an Infectious Human Endogenous Retrovirus. PLoS Pathogens 3(1): e10 doi:10.1371/journal.ppat.0030010
Retrovirus genomes integrate into the genomes of host cells. If the target cells of a particular retrovirus include germ-line cells, e.g., sperm or egg cells, then retroviral genomes can be inherited like cellular genes. So-called endogenous retroviruses have accumulated throughout evolution in the genomes of many organisms, including humans. While all known endogenous retroviruses of modern humans are unable to replicate as retroviruses, the human genome represents a fossil record of ancient retroviruses that once infected our ancestors. In this study, a collection of "dead" endogenous retroviral genomes in modern human DNA was used to deduce the approximate sequence of an ancestral retrovirus, human endogenous retrovirus (HERV)-K, that is now thought to be extinct. A pseudo-ancestral HERV-K DNA sequence was synthesized and used to produce viral proteins and RNA that could reconstitute the HERV-K replication cycle. Thus, the replication and biology of a once-extinct retrovirus can now be studied in the laboratory. Interestingly, reconstituted HERV-K replication experiments, and comparison of the reconstituted HERV-K DNA sequence with the dead HERV-Ks in modern human DNA, suggests that HERV-K may have been extinguished in humans in part by host defenses that induce mutation of retroviral DNA and that the reconstitution of the pseudo-ancestral HERV-K reversed these changes.
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 5alpha 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.
A characteristic that is unique to retroviruses is their propensity to integrate their genomes into host-cell DNA as an essential part of their replication cycle. Thus, if the target cell population of a given retrovirus includes germ cells or their progenitors, retroviral genomes can be inherited in a Mendelian manner as so-called "endogenous" forms (see  for review). Indeed, endogenous retroviruses have accumulated over time in the genomes of many organisms and are extraordinarily common in mammals, comprising approximately 8% of human DNA . Nonetheless, while some avian, murine, and primate species harbor replication-competent retroviruses within their genomes, intact retroviruses are relatively infrequent and almost all endogenous retroviruses are obviously defective due to the presence of stop codons and frameshifts in one or more genes.
Among the numerous families of defective human endogenous retroviruses (HERVs) found in modern human DNA, the human mouse mammary tumor virus-like 2 (HML-2) subfamily of HERV-K proviruses is of special interest. Even though replication-competent forms of HERV-K(HML-2) have not been found, some proviruses were deposited in the human genome after speciation and represent some of the youngest HERVs known [3-6]. Also, occasional reports link their expression with human disease . The age of an endogenous provirus can be roughly estimated by comparing sequence of the two long terminal repeats (LTRs). At integration, the two proviral LTRs should be identical, but during host DNA replication, each LTR independently accumulates mutations as a function of age, and it is estimated that one difference between two LTRs should occur every approximately 200,000 to 450,000 y. Several HERV-K(HML-2) proviruses have been identified in human DNA that have less than five differences between the two LTRs, suggesting deposition perhaps less than 1 million y ago [3-6]. HERV-K(HML-2)-related proviruses are found only in Old World primates genomes, and many are unique to humans, with nonhuman primate genomes containing empty preintegration sites at orthologous loci. Compellingly, polymorphism exists in humans with respect to the presence or absence of proviruses at some HERV-K integration sites, indicating insertion relatively recently in human evolution [3-6]. Furthermore, many of the younger HERV-K(HML-2) proviruses contain a subset of open reading frames (ORFs) with a few or no mutations [3,6,8]. However, all known HERV-K proviruses are replication defective.
There are several ways in which a defective provirus can proliferate in a host's genome, including via exogenous infection events following complementation in trans, where functional proteins are supplied by other endogenous or exogenous viruses. Alternatively, for some retroelements, envelope-independent retrotransposition can occur in cis, where an element copies itself and inserts into a new genomic locus within the same cell, forgoing the normal extracellular phase of the retroviral life cycle. Defective proviruses can also be proliferated as a result of long interspersed element retrotransposition . However, most HERV-K(HML-2) replication appears to have been a consequence of autonomous infection by extracellular virions [10,11]. This conclusion is based on the comparatively low number of stop codons and ratio of nonsynonymous to synonymous changes (dN/dS) in HERV-K ORFs, indicating a purifying selection on all proteins. Notably, this finding holds for HERV-K Env , which should be required for replication that includes an extracellular step but not for any other mode of provirus proliferation.
Ancient retroviruses are of interest, in part because they likely imposed selective pressure on host defenses in human ancestors. Indeed, the tripartite motif (TRIM) 5alpha and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 3 proteins that provide part of the host defense against modern retroviruses have been under positive selection for much of primate evolution [12-16]. As a retrovirus that appears to have replicated in the ancestors of modern Old World monkeys, apes, and humans, HERV-K may be partly responsible for this pressure. Moreover, it is conceivable that HERV-K exists today in an undetected replication-competent form in rare humans . However, no studies of the virology or pathogenic potential of this ancient human virus have been possible because a contemporary, replication-competent HERV-K strain has not been identified and may not exist at all.
Despite some functional degradation due to mutation during deposition or during human DNA replication, HERV-K(HML-2) proviruses that have been deposited in human DNA in the past few million years should be reasonably well preserved and have relatively few inactivating mutations. Indeed, various studies have shown that individual proteins from certain HERV-K proviruses can function in vitro [17-25]. We reasoned that it might be possible to resurrect HERV-K(HML-2) in replication-competent form using proviruses that are thought to have most recently entered the human genome as a template. Therefore, we constructed a HERV-K strain whose genome sequence is a consensus of a subset of HERV-K(HML-2) proviruses. Importantly, we demonstrate that all viral proteins necessary for viral replication encoded by this provirus are functional and that proteins and genomes based on the reconstructed HERV-K(HML-2) viral genome can be used to generate infectious exogenous retrovirus particles.
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