The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation

Abstract

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn’t recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3–6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3–6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3–6 hairpin than the wild-type hairpin. The SΔ1Δ3–6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30–37 days, while mice infected with SΔ1Δ3–6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV “flip and flop” telomeres are arranged.

Polinton-like Viruses Associated with Entomopoxviruses Provide Insight into Replicon Evolution

“…Similarly, MySEV encoded a tyrosine recombinase (Figure 4C) and a primase-helicase (Figure 4D) that were closely related to those of CBEV_PLV1. While these MySEV components were encoded in separate regions of the MySEV genome, poxviruses have been hypothesized to initiate replication out of hairpin structures, referred to as telomeres, that occur within the TIRs(Shenouda et al 2022). Tyrosine recombinase activity on these ends could be a mechanism for genome dimer resolution, as occurs in bacterial genomes, plasmids, and some bacteriophages (Castillo et al 2017; Haenebalcke and Haigh 2013).…”

Single Dose of Recombinant Chimeric Horsepox Virus (TNX-801) Vaccination Protects Macaques from Lethal Monkeypox Challenge

“…Recombinant chimeric HPXV (rcHPXV) and vaccinia (rVACV) were utilized as vaccines. The methods for the generation of rcHPXV and rVACV have been previously described [ 19 , 23 ]. The rVACV genome sequence was deposited into GenBank (Accession # MN974381).…”

Poxvirus Recombination

“…Finally, reactivation reactions have also been used to reconstruct and reactivate poxviruses using DNA fragments obtained through wholly synthetic approaches. The first such example was a 220 kbp horsepox virus [204], but synthetic copies of VACV strains MVA [205], and Acambis 2000 [206], have since also been constructed in similar ways. An advantage of these methods is the flexibility that gene synthesis offers, and as the cost of gene synthesis continues to decline [207], and interest in more complex and personalized recombinants grows, may displace many of the methods devised to date.…”

“…An advantage of these methods is the flexibility that gene synthesis offers, and as the cost of gene synthesis continues to decline [207], and interest in more complex and personalized recombinants grows, may displace many of the methods devised to date. It makes possible the construction of mutants that would be difficult to produce in any other way [206]. Of course, the technology is not without controversy.…”