Retroviruses comprise a distinct group of enveloped RNA viruses that replicate by reverse transcribing their RNA genomes to form a DNA copy within a viral core particle. The DNA copy, or cDNA, is then integrated into the host genome. The integrated proviral DNA is transcribed by RNA polymerase II (Pol II) to produce polyadenylated mRNAs that are translated into viral proteins and also packaged into assembling core particles in the cytoplasm or at the plasma membrane. Core particles acquire a host-derived envelope as they bud out of the cell. The membrane of retroviral virions fuses with the membrane of new host cells, and the replication cycle begins again (Fig. (Fig.1).1). Retroviruses are obligate parasites with small genomes and a complex mode of replication; thus, they are reliant on a multitude of host factors for replication. At the same time, mammalian cells have evolved a variety of mechanisms to impede retroviruses, which are potentially pathogenic or mutagenic to their hosts. Significant progress toward identifying mammalian host factors that regulate the activities of retroviruses has been made in recent years. A number of dominant retroviral resistance factors, including the APOBEC3 family of cytosine deaminases, the mouse Fv1 restriction factor, and the primate antiviral factor TRIM5α, have been uncovered using genetic approaches (reviewed in references 7 and 49). In addition, a diverse collection of recessive genes that promote replication at a variety of steps in the retroviral life cycle have been identified through the analysis of biochemical and two-hybrid interactions and dominant-negative mutants (reviewed in references 47 and 48). This body of work has deepened our appreciation of the complexity of the host-retrovirus relationship and illustrated how much remains to be understood about the intricate interplay between host and pathogen. In this review, we explore the value of using a simple model organism to systematically identify functional orthologs of host factors involved in retroviral replication.
A facile approach to the identification of mammalian genes that participate positively or negatively in retroviral propagation is first to identify genes that regulate retrovirus-like transposons in a model organism and then to test the effect on retroviral replication of mutating or reducing the expression of the corresponding mammalian protein. The development of tools to specifically reduce the expression of individual genes through RNA interference in many mammalian species has dramatically enhanced the feasibility of this approach. Retrovirus-like transposons are ubiquitous in eukaryotes and constitute a significant percentage of the host genome, from 3% of the genome of the budding yeast Saccharomyces cerevisiae to approximately 8% of the human genome (12, 36). While retrovirus-like transposons are not pathogenic, they are potent insertional mutagens. Many of the steps in transposition, with the notable exception of viral-particle budding and infection of new cells, are analogous to steps involved in the replication of retroviruses (Fig. (Fig.1).1). Consequently, the Ty1 and Ty3 retrovirus-like transposons in S. cerevisiae have been studied extensively as models to explore the influence of the host cell on retroviral propagation (71, 87, 106). The stable haploid phase of growth, the availability of genetic and genomics tools, and the feasibility of biochemical studies of S. cerevisiae are some of the features that permit rapid identification of host factors and analysis of their effects on the replication of retrovirus-like elements.
Retroviruses and retrovirus-like transposons consist of terminal direct-repeat sequences known as long terminal repeats (LTRs) flanking a central coding region, which at a minimum consists of gag, pol, and, in some cases, prt genes. The gag gene encodes the structural protein or proteins necessary to form the viral core particle or the virus-like particle (VLP). The pol domain encodes reverse transcriptase (RT) and integrase (IN) enzymatic activities and sometimes protease (PR) activity, if PR is not encoded by a separate prt gene. Retroviruses and retrovirus-like transposons, also known as LTR retroelements, can be divided into three major families on the basis of their amino acid sequences in RT: the retroviruses (Retroviridae), the Ty3/gypsy/BEL family (Metaviridae), and the Ty1/copia family (Pseudoviridae) (52). Retroviral genomes harbor an additional open reading frame (ORF) called env, which encodes an envelope glycoprotein that is incorporated into the cell-derived lipid bilayer of the retroviral virion when it exits the cell. The presence of a functional env gene is correlated with the infectivity of retroviruses, and acquisition of env is thought to be a key step in the evolution of retroviruses from LTR retrotransposons (63). While most members of the Ty3/gypsy/BEL and Ty1/copia families lack both an env gene and an extracellular phase of replication, the two families do include several examples of elements that have obtained env-like ORFs (52), and the Drosophila metavirus gypsy has been shown to be an infectious retrovirus (97). Regardless of whether they carry an env gene, metaviruses, such as Ty3, are more closely related to retroviruses, and they have a common structural organization of the pol domain. Pseudoviruses, such as Ty1, comprise the most ancient family of LTR retroelements.
The identification of conserved host genes that inhibit or contribute to Ty1 and/or Ty3 retrotransposition provides candidates for genes that influence retrovirus replication. Recent genetic screens for host factors that control Ty1 or Ty3 retrotransposition have identified large sets of genes involved in a variety of basic cellular processes (4, 50, 56, 90). Some regulatory themes are evident among these collections of host factors and restriction factors, such as regulation through RNA-processing factors and DNA damage-signaling/repair proteins. The utility of the yeast model systems is emphasized by the recent identification of a small number of genes involved in RNA processing and DNA repair as regulators of both retrovirus replication in mammalian cells and Ty1 and/or Ty3 elements in yeast (67, 110, 111). Moreover, the human antiviral protein, APOBEC3G, when expressed in yeast potently inhibits Ty1 retrotransposition by a mechanism similar to that by which human immunodeficiency virus type 1 (HIV-1) replication is restricted in human cells (40, 92). These recent findings suggest that not only the mechanisms of retroelement replication, but also the various means by which retroelements interact with their hosts, can be conserved in eukaryotes.