Genetic diversity of koala retroviral envelopes

Abstract

Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

Figure 1

Schematic representation of the KoRV proviral genome showing positions of primer pairs used to clone KoRV envelope variants and defective KoRV envelopes. Diagram not to scale. *Primers used in previous studies [3,14].

Figure 2

(A) Genomic organization of the defective GFP expressing retroviral vector. The long terminal repeats (LTRs) including U3, R, and U5 regions are derived from Moloney murine leukemia virus (Mo-MLV). The defective GFP genome is integrated into the genome of 293T-GFP cells and can be rescued after co-culture with koala PBMCs harboring infectious KoRVs; (B) Experimental design used to determine whether KoRV-B envelopes can pseudotype the KoRV-A genome. Green-filled circles indicate GFP expressing cells. Infectious KoRV-B enveloped viruses obtained by co-culturing 293T-GFP with koala PBMCs were used to infect KoRV-A-resistant MDTF-THTR1 cells. Wildtype and pseudotyped viruses in the supernatant of infected MDTF-THTR1 cells were subsequently used to infect MDTF-PiT1 and MDTF-THTR1 cells.

Figure 3

(A) KoRV-A, B, E, and F envelope proteins differ mainly in the VRA region. Alignment of the amino terminus of KoRV-A,-B,-E and -F (corresponding to residues 86–230 of KoRV-A) envelope proteins. Residues that differ among this envelope segment are highlighted in yellow; (B) Predicted KoRV-F residues comprising the amino terminal region of the envelope protein aligned to the corresponding regions of KoRV-C, D, and E and F residues (residue numbers correspond to KoRV-A envelope residues 86–230). The putative VRA and VRB regions are underlined with residues that differ among the four isolates highlighted in yellow.

Figure 4

(A) The VRA region of KoRV-F is related to VRA regions of KoRV-C and PC010 KoRV envelope [16]. Envelope residues corresponding to KoRV-A envelope residue numbers 86–230 are aligned. VRA and VRB regions are underlined. Residues that differ among KoRV-E, F and PC010 are highlighted in yellow; (B) Schematic representation of the sites where the 18 nucleotide repeats are positioned within the U3 enhancer regions (dark grey) of KoRV-B and KoRV-F. The grey bar represents the tandem repeat of the 18-nucleotide insertion, with KoRV-A containing a single copy, KoRV-B four copies and KoRV-F five copies. The 18 nucleotides are boxed.

Figure 5

KoRV-E and KoRV-F are not vertically transmitted. All koalas are KoRV-A positive. Male and female koalas are denoted as square and circle symbols, respectively, in the family tree. KoRV-E positive koalas are denoted as $\text{E+}$ and KoRV-F positive koalas as $\text{F+}$.

Figure 6

Mechanism of KoRV enveloped retroviral transmission in koalas infected with KoRV-A (represented as koalas within blue circles), KoRV-B (red encircled koalas) and/or KoRV-F (koalas within purple circles). The small circles represent KoRV viral particles composed of KoRV-A, B, or F envelopes (blue, red or purple, respectively) and the folded lines within the circles represent the KoRV-A, KoRV-B or KoRV-F genomes (red, blue or purple lines, respectively). KoRV-A positive animals are resistant to superinfection by KoRV-A (top most figure) but the KoRV-A genome (blue) can be transmitted in particles bearing KoRV-B (red) or KoRV-F (purple) envelopes.