Classification of PERVs
Endogenous Retrovirus (ERVs) discovery occurred in the late 1960s and early 1970s (Weiss, 2006). The first reports on Avian Leukosis Virus (ALV), Murine Leukaemia Virus (MLV), Murine Mammary Tumor Virus (MMTV) and PERVs were almost at the same time (Garcia-Montojo, Doucet-O’Hare, Henderson, & Nath, 2018). Breese et al. first described PERVs in 1970 as virus-like particles, same as those seen in the baby hamster kidney (BHK-21) cell line infected with MLV (Breese, 1970). And according to the morphological properties, some investigators speculated that these viral particles belonged to the Type C Virus (Armstrong, Porterfield, & De Madrid, 1971). In 1974, George et al. further characterized the biochemical and immunological properties of the viral particles and demonstrated that it was a typical type C virus (Todaro, Benveniste, Lieber, & Sherr, 1974).
Two sets of human-tropic PERVs were first reported in 1997. By using the 3’ Rapid Amplification of Cloned Ends (3’RACE), the Weiss team obtained two classes of sequences (PERV-A and PERV-B) from the PK15 and human embryonic kidney 293 cells (HEK-293) infected with PERVs (Le Tissier, Stoye, Takeuchi, Patience, & Weiss, 1997). The distinct third class of PERV is PERV-C, isolated from a swine lymphoma infected only with pig cells (Akiyoshi et al., 1998; Takeuchi et al., 1998). The PERV A and B are found to be polytropic, and the PERV C is ecotropic only infecting pig cells (Wilson, Wong, VanBrocklin, & Federspiel, 2000).
In 2001, in addition to the PERV-A, -B, and -C, classified together as gamma genera of retroviruses (gamma-1), Patience et al.detected the DNAs of four novel groups of gamma-retrovirus (gamma-2 to gamma-5) and four novel groups of beta-retrovirus (beta-1 to beta-4) in the genomes of domestic pigs (Patience et al., 2001). And so far, only the gamma-1 PERVs remain replication-competent as an active provirus.
PERV-A, -B and -C subfamilies are mainly different in env gene sequence which encodes envelop protein, while the other genes encoding internal proteins, including gag and pol , are highly conserved among the three subfamilies. The PERV-A and PERV-C could form a hybrid virus named as PERV-A/C through a recombination of theenv gene (Denner, 2008). As one part of the env gene is originated from PERV-A, the recombinant PERV-A/C virus can infect human cells. And associated with alterations in the LTR sequences, the recombinant virus exhibits an increased replication capacity (Denner, Specke, Thiesen, Karlas, & Kurth, 2003). Fortunately, by using PERV-C free pigs, the risk of PERV-A and PERV-C recombination in xenotransplantation can be eliminated.
Molecular structure of PERVs
PERVs are enveloped-linear ssRNA retroviruses containing two copies of the genomic RNA. Infectious PERVs (PERV-A, -B, and -C) belong to the Retroviridae family, Orthoretrovirinae subfamily, Gammaretrovirus genus. The transcript of PERVs has three open reading frames (ORFs) which respectively encode structural proteins (Gag), polymerases (Pol) and glycoproteins (Env) (Fig.1a).
The Gag proteins consist of three major domains: the matrix (MA), the capsid (CA), and the nucleocapsid (NC). The MA domain initially binds to the membranes and detaches from the host cell during budding.
The CA domain mediates the crystal lattice-forming protein interactions in capsids. And the NC domain is responsible for the packaging of the viral RNA genome (Denner & Tönjes, 2012; Pornillos & Ganser-Pornillos, 2019).
In PERVs, the pol gene encodes the protease (PR), the integrase (IN), and the reverse transcriptase (RT). The PR can be auto-activated to cleave Gag and Pol at specific sites to initiate PERV maturation (Blusch, Seelmeir, & von der Helm, 2002). The RT is a key enzyme in viral propagation, generating complementary DNAs (cDNAs) from the PERV ssRNA templates. With the help of the IN, the PERV cDNAs can be integrated into the host genome, from which the new viral copies can be generated via host transcription (Łopata, Wojdas, Nowak, Łopata, & Mazurek, 2018).
The Env protein plays a critical role in the entry of PERV particles into the host cells during viral replication cycle. Env protein is synthesized as a trimeric precursor in the endoplasmic reticulum, which is subsequently cleaved into a surface subunit (SU) and a transmembrane subunit (TM) by a cellular furin-like PR(Fig.1b). The SU subunit contains a receptor-binding domain (RBD) which binds to a specific receptor on the target cell. Through the hydrophobic domain, the TM subunit is buried in the lipid bilayer of the viral particle. And the cytoplasmic tail of the TM subunit contains an R peptide, which will be cleaved during virion maturation to activate the fusion capacity (Bobkova, Stitz, Engelstädter, Cichutek, & Buchholz, 2002) (Fig.1c). The CxxC motif is localized at the C-terminus of the SU and covalently links the CXnCC motif on the TM to form an inter-subunit disulfide bond (Johnson, 2019). The disulfide bond anchors the SU subunit to the surface of PERV particles (Fig.1b, c).
In the proviral form of PERVs, the coding sequences of gag ,pol , and env are flanked by a 5’ and a 3’ long terminal repeats (LTRs). And each LTR contains a unique 3 (U3), repeat region (R), and unique 5 (U5) regions in 5’ to 3’ direction: U3-R-U5 (Denner, 2021). The transcription of PERVs starts at the U3–R junction in the 5ʹ LTR, and ends at the R–U5 junction in the 3ʹ LTR. LTRs play an essential role in the replication cycle of PERVs (Łopata et al., 2018). Moreover, LTRs possess promoters, enhancers and multiple transcription factor binding sites (Jung et al., 2013), which drives the transcription of PERVs within the host genome.