Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions

Abstract

Viruses of the subfamily Orthoretrovirinae are defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and "fossil" endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions.

Keywords: bioinformatics; endogenous retrovirus; hybrid capture; iatrogenic transmission; orthoretrovirus; retroviral receptor; retroviral transmission; taxonomy.

FIG 1

(A) An integrated double-stranded DNA provirus (yellow) of a simple orthoretrovirus within the host genome (gray) is shown. The long terminal repeats (LTRs) are at both the 5′ and 3′ ends of the provirus and flank the retroviral gag, pol, and env coding regions. Regions coding for enzymes and other proteins are shown with font colors corresponding to their depiction in panel B. (B) Schematic drawing of a simple orthoretrovirus. All orthoretroviruses have three component parts: (i) the RNA genome, shown in yellow; (ii) internal proteins, shown in red, including internal structural proteins (Gag) as well as the viral enzymes, including the reverse transcriptase (Pol), which makes a DNA copy of the RNA genome which will be integrated into the host cell genome; and (iii) the envelope proteins, shown in blue. Env consists of two components: the TM moiety is embedded in the membrane (depicted in gray and white) of a host cell and is incorporated into the virion during the budding process, and the surface glycoprotein SU forms the knobs and is the part of the virus that binds to receptors on susceptible cells of the host.

FIG 2

Mechanisms of XRV and ERV transmission. XRVs are transmitted horizontally (red arrows) either in utero or via infected blood, feces, urine, milk, or saliva. After an XRV invades the germ line, ERVs (green arrows) may be amplified by superinfection or retrotransposition in the host germ line. ERVs that remain infectious can potentially infect naive members of the species. (Koala photographs courtesy of Tad Motoyama [Los Angeles Zoo, Los Angeles, CA].)

FIG 3

Classification of the Retroviridae into subfamilies and genera. The Retroviridae family is not assigned to an order.

FIG 4

Organization of the three classes of transmembrane (TM) proteins found in orthoretroviruses and the genera of retroviruses in which they are found (21). The immunosuppressive domain (ISD) and the CX₆CC cysteine motif with disulfide bonds are depicted for TM classes 1 and 2. In class 3, the alternate cysteine motif CX_nC and the membrane-proximal external region (MPER) are present, whereas the transmembrane region (TR), cytoplasmic tail (CT), and fusion peptide are variably positioned within the TM domain but invariably present in all TM proteins.

FIG 5

Summary of koala retrovirus (KoRV) statuses within a pedigree of 17 northern Australian koalas kept at the Los Angeles Zoo. Red koala symbols denote individuals positive for KoRV-A (which is ubiquitous in U.S. zoos), whereas the blue koala symbols denote individuals positive for KoRV-B (which is not ubiquitous) and KoRV-A. Dead joeys ejected from the maternal pouch are depicted within squares; each was positive for KoRV-B. Note that the pattern is consistent with maternal transmission of KoRV-B.

FIG 6

Geographic distributions of species harboring KoRV, GALV, and related retroviruses. Red shading indicates the historical distribution of the white-handed gibbon (Hylobates lar); five GALV strains have been isolated from this species. The range of several species of Mus (Mus cervicolor, Mus dunni, and Mus musculus), carrying McERV (296), MDEV (297), and MmERV (298), respectively, is indicated in blue; Mus was originally proposed as the source of GALV. The green region approximates the range of koalas (Phascolarctos cinereus) in eastern Australia, many of which carry KoRV-A. The distribution of KoRV-B among wild koalas has yet to be determined. Orange shading indicates the range of Melomys burtoni and the two GALVs identified in this species: MbERV and MelERV. The broken brown line and shading indicate the overall distribution of the genus Melomys. Wallace's Line (dashed black line) is a biogeographic (deep water) barrier separating the terrestrial fauna of Southeast Asia, including western Indonesia, from that of eastern Indonesia, New Guinea, and Australia. (Photographs courtesy of Serge Morand and CeroPath [rodents], Tilo Nadler [gibbon], Daniel Zupanc [koala], and Neil Furey [bats].)

FIG 7

Summarized phylogenetic relationships of KoRVs, GALVs, and related gammaretroviruses. Rodent retroviruses are shown in red, bat retroviruses are shown in green, retroviruses within the GALV clade are shown in blue, KoRVs are shown in yellow, and related gammaretroviruses from other taxa are shown in gray. Lineages under episodic diversifying selection within the GALV-like viruses are marked with red asterisks.

FIG 8

Phylogeny of retroviral envelope sequences (left) and gene sequences (right) of the host receptors used by viruses. Phylogenies were inferred using the neighbor-joining method implemented in MEGA v7.0.18 (299). Human gene sequences were used to generate the tree of receptor gene sequences, with slc01a2 (28) used as the outgroup. ALV-J was used as the outgroup for the virus phylogeny, which includes the viruses listed in Table 2. Bootstrap values of ≥80% for 10,000 replicates are shown. Colored lines connect each virus to the gene for the receptor that it uses. GenBank accession numbers for gammaretroviruses are as follows: 10A1-MLV, M33470; A-MLV, AF411814; X-MLV, m59793; P-MLV, KJ668271; E-MLV, KJ668270; M813, AF327437; HEMV, AY818896; FeLV-B, K01209; FeLV-A, AF052723; FeLV-C, U58951; GALV, KT724048; KoRV-A, AF151794; KoRV-B, NC_021704; HERV-W, AH013146; REV, KU204702; RD-114, NC_009889; BaEV-M7, NC_022517; SRV, U85505; ALV-J, KP284572; BLV, M35242; and HTLV-I, AB600229. GenBank accession numbers for receptor genes (human gene product) are as follows: XPR1 (ND), NM_004736; slc20a1 (PiT-1), NM_005415; slc20a2 (PiT-2), NM_001257180; slc19a2 (THTR1), NM_006996; slc49a1-4 (FLVCR1), NM_014053; slc5a3 (SMIT1), NM_006933; slc7a1 (MCAT), NM_003045; slc1a5 (ASCT1), NM_005628; slc1a4 (ASCT2), NM_003038; slc01a2, NM_134431; and slc2a1, NM_006516.

FIG A1

Methods used to determine whether a given sequence is present, to characterize full genomes by hybridization capture (HC), and to characterize ERV integration sites by inverse or linker-based PCR approaches. (A) Southern blotting and pan- or broad-specificity PCRs are shown as examples of low-throughput methods. Southern blotting will provide information on whether homologous sequences are present in a given DNA extract but not whether the sequences are closely related, divergent, or a mixture of both. PCR-based approaches using conserved retroviral gene regions can produce limited sequence information from single or low-copy-number ERVs. However, if a high-copy-number ERV that has diversified is being amplified, amplification may fail for some loci due to primer mismatch, while the amplification of many loci at once will result in many polymorphisms at different positions in the sequence. Cloning and sequencing of the PCR product would be needed to sequence distinct ERVs. To determine sequence diversity comprehensively, high-throughput sequencing (HTS) methods must be used. (B) Hybridization capture. HTS libraries are built from fragmented genomic DNA or cDNA. The sequencing platform employed is largely irrelevant if the correct sequencing platform adaptors are used in building the libraries (and correct blocking oligonucleotides are used during capture). Baits for enriching the target sequence of interest can be generated by PCR, generally involving large amplicons (depending on the target) and fragmenting them to avoid having baits that are much larger than target library molecules (300). Alternatively, the baits can be synthesized much like the synthesis of PCR primers and can be made of DNA or RNA. Recently, entire genomes have been used to enrich highly contaminated samples for genomic DNA, for example, using elephant DNA to enrich woolly mammoth DNA from fossils (301). In some cases, microarrays, such as virus microarrays, may be used as baits (302, 303). In the figure, solution-based hybridization capture is shown. Regardless of strategy, the resulting captured library is massively enriched for target sequences of interest, while nontarget sequences are washed away prior to HTS. In addition to target sequence enrichment, the sequences flanking the target will also be captured as library sequences extending just beyond the end of the bait or even further are also captured by the annealing of overlapping sequences, or “CapFlank” (219). (C) Additional methods for enriching ERV integration sites. By fragmenting genomic DNA, circularizing the fragments in a ligation step, and then applying inverse PCR to the resulting circular DNA, one can extend known sequences into flanking unknown sequences based on PCR primers anchored entirely within the known sequence (inverse PCR). Linker-based approaches use a somewhat similar strategy; however, instead of using two primers based on a known sequence, a known linker sequence is ligated to the ends of fragmented DNA, and one PCR primer is located on the linker sequence, extending across the unknown sequence toward the other primer, which is based on the known sequence. Sequencing of the amplicons can then be performed by low- or high-throughput methods.