This VVΔEΔK+RhTRS1 replication defect in PRO1190 cells may be due to incomplete inhibition of PKR in these cells by RhTRS1. To test this hypothesis, we generated PRO1190 cells stably expressing either a PKR-specific shRNA (PRO1190-PKR kd), which resulted in a 56% reduction of PKR expression, or a scrambled control shRNA (PRO1190-ctrl kd) ( Fig. 1B ). Similarly, PKR-specific RT-qPCR demonstrated a 60% reduction in PKR mRNA from PRO1190-PKR kd cells relative to PRO1190-ctrl kd cells (2578 copies or 6503 copies PKR/ng total RNA respectively) but little difference between PRO1190 and PRO1190-ctrl kd cell PKR levels ( Fig. S1 ). In PRO1190-PKR kd cells, VVΔEΔK+RhTRS1 replication was almost completely rescued to VV-βg levels ( Fig. 1C ). We detected a similar increase in VVΔEΔK+RhTRS1 replication after transiently transfecting PRO1190 cells with a siRNA specific for PKR, but not a control siRNA (data not shown). Sequence analysis of PRO1190 PKR identified three non-synonymous mutations relative to a previously reported AGM PKR (GenBank # EU733254) that is sensitive to RhTRS1 ( Fig. S2 , [20] ). Interestingly, one of these single nucleotide variants is heterozygous in PRO1190 cells, and changes a residue (T577M) that is evolving under positive selection in primates [12] . Although additional studies will be needed to determine whether one or more of these PRO1190 PKR polymorphisms is responsible for increased resistance to RhTRS1, the results shown in Fig. 1 demonstrate that the block to VVΔEΔK+RhTRS1 replication in PRO1190 cells is mediated by PKR.

(A) Titers of the indicated viruses produced 48 hpi (MOI = 0.1) of PRO1190 (gray bars) or BSC40 cells (black bars), measured using HFF+TRS1. (B) Immunoblot of lysates of PRO1190 stably transduced with lentiviral vectors expressing control (left) or PKR-specific (right) shRNAs, probed with anti-PKR or anti-actin antibodies. (C) Titers of the indicated viruses 48 hpi (MOI = 0.1) in either PRO1190+ctrl shRNA (grey bars) or PRO1190+PKR shRNA (black bars), measured using BSC40 cells. Results are represented as means +1 STD.

The rhesus cytomegalovirus (RhCMV) PKR inhibitor TRS1 (RhTRS1) can block PKR activation and rescue replication of a vaccinia virus mutant lacking the PKR inhibitor E3L (VVΔE3L+RhTRS1) in several African green monkey (Chlorocebus aethiops, AGM) cell lines [20] . However, we discovered that a recombinant vaccinia virus expressing RhTRS1 and lacking both of the known vaccinia PKR antagonists, E3L and K3L, (VVΔEΔK+RhTRS1) produced 100 to 1000-fold less virus in AGM-derived PRO1190 cells relative to VV-βg (which contains both E3L and K3L) although it replicated almost as efficiently as VV-βg in AGM-derived BSC40 cells ( Fig. 1A ). Thus, RhTRS1 varies in its ability to support VVΔEΔK replication in different AGM cells.

Rhtrs1 amplification expands virus tropism to human and rhesus macaque cells

To determine whether VVΔEΔK+RhTRS1 could adapt to overcome the PKR-mediated block to replication in PRO1190 cells, we utilized a system of experimental evolution. We infected PRO1190 cells with VVΔEΔK+RhTRS1 at a low multiplicity of infection (MOI = 0.1). 48 hours post infection (hpi) we lysed the infected cells, titered the resulting virus, and infected new PRO1190 cells, repeating this cycle multiple times. After four passages we observed a 10- to 100-fold increase in viral replication that remained stable for at least three subsequent passages in each of three independent lineages (Fig. 2). We next performed a competition assay to assess the relative fitness of the passaged virus in comparison to the initial VVΔEΔK+RhTRS1 virus. We co-infected PRO1190 cells with either VVΔEΔK+RhTRS1 or VV-A, both of which express eGFP, and the same competitor, VVΔE3L+RhTRS1, which expresses β-gal (MOI = 0.1 for each virus). Virus produced 48 hpi was titered on permissive BSC40 cells and the specific progeny viruses were enumerated by detecting β-gal (VVΔE3L+RhTRS1) and eGFP (VVΔEΔK+RhTRS1 and VV-A) expression in the plaques (Fig. S3). VVΔEΔK+RhTRS1 replicated ∼3-fold better than VVΔE3L+RhTRS1, whereas VV-A replicated 290-fold better than VVΔE3L+RhTRS1, confirming that serial passage through PRO1190 cells increased the fitness of passaged viruses approximately 100-fold relative to the initial VVΔEΔK+RhTRS1.

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larger image TIFF original image Download: Figure 2. Experimental evolution of VVΔEΔK+RhTRS1 increases replication fitness in PRO1190 cells. PRO1190 cells were initially infected with VVΔEΔK+RhTRS1 (MOI = 0.1). Virus was harvested at 48 hpi, titered on BSC40 cells, and used to infect fresh PRO1190 cells and the process was repeated. Three independent infections resulted in an ∼10-fold to 100-fold gain of replication fitness in PRO1190 that was evident by passage four. https://doi.org/10.1371/journal.ppat.1004002.g002

Since the passaged viruses replicated more efficiently in a minimally permissive cell line, we investigated the ability of the passaged viruses to replicate in primary cells from more divergent primates. We have shown that RhTRS1 does not inhibit human or rhesus PKR in the context of VVΔE3L [20]. To determine whether the adaptations that evolved during serial passage in PRO1190 cells affected the virus species tropism, we infected primary human foreskin fibroblasts (HFF) or primary rhesus fibroblasts (RF) with VVΔEΔK+RhTRS1, each of the three passaged virus pools or VV-βg at an MOI of 0.1 (Fig. 3). As expected, VVΔEΔK+RhTRS1 replicated poorly and VV-βg replicated efficiently in each cell type. Remarkably, all three passaged pools replicated between 1000- to 10,000-fold better than VVΔEΔK+RhTRS1 in both HFF (Fig. 3, center) and RF (Fig. 3, right). For each pfu of VV-A, VV-B and VV-C used to infect the cells, 5.2, 1.9, and 6.3 pfu of progeny emerged from HFF and 40.8, 7.3, and 3.7 emerged from RF, respectively, suggesting that these viruses were sufficiently well adapted to enable continuous propagation in these cells (see below). However, these virus pools still replicated 10- to 100-fold better in PRO1190 than in either human or rhesus cells (Fig. 3, left). Thus, adaptation of VVΔEΔK+RhTRS1 in minimally permissive AGM fibroblasts also provides a substantial replication benefit in human and rhesus cells expressing distantly related PKR proteins. To elucidate the underlying mechanism for this gain of fitness, we harvested DNA from passage 7 viruses for genetic analyses and then passaged the viruses once more in PRO1190 to generate viral stocks for biochemical and infectivity analyses.

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larger image TIFF original image Download: Figure 3. Adaptation in minimally-permissive PRO1190 cells expands the species tropism of VVΔEΔK+RhTRS1. PRO1190 (left), HFF (center), or RF (right) were infected with the indicated viruses (MOI = 0.1). Lysates were harvested 48 hpi and titered on BSC40 cells. Data are represented as means +1 STD. https://doi.org/10.1371/journal.ppat.1004002.g003

Gene amplification as a mechanism of rapid adaptation in vaccinia virus has been well documented [17], [18], [21]. To determine whether gene amplification could account for the broadly improved replication of passaged VVΔEΔK+RhTRS1 we performed paired-end Illumina based deep sequencing (Short Read Archive #SRP033208). Based on read depth, we detected duplication of the rhtrs1 locus in all three passage 7 virus pools but not in VVΔEΔK+RhTRS1 (Fig. 4A). Each of the passaged pools contained between 1.4 and 1.9 copies of rhtrs1 per genome, although these numbers reflect averages of a heterogeneous population of viral genomes. Confirming this estimate of rhtrs1 copy number, the frequency of reads in which we detected a recombination site near the rhtrs1 locus increased as a percentage of total reads in viruses predicted to have more copies of rhtrs1 (Fig. 4B).

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larger image TIFF original image Download: Figure 4. Rhtrs1 copy number variation relative to VVΔEΔK+RhTRS1. (A) Relative read depth of Illumina sequencing reads in the passaged virus pools normalized to VVΔEΔK+RhTRS1. The graph is centered on the rhtrs1 locus (grey bar). (B) Copy number of RhTRS1 (rescaled so that parental strain = 1) plotted against breakpoint read frequency (per million mapped reads) demonstrates agreement between each independent measure of RhTRS1 duplication. https://doi.org/10.1371/journal.ppat.1004002.g004

We used PCR to confirm that the rhtrs1 locus was amplified, using externally directed primers specific to rhtrs1 that only amplify a product if there is a tandem duplication of the gene (Fig. S4A). We detected 3 kb products in all three virus pools, and 2.3 kb and 1.8 kb products only in the VV-A virus pool (Fig. S4B). We were unable to obtain enough of the 2.3 kb band for further analysis, but we did characterize the 3 kb and 1.8 kb products by Sanger sequencing. The larger product was identical in all three passaged virus pools, and represents a recombination between the vaccinia virus gene L5R upstream of rhtrs1 with J2R downstream of rhtrs1. In the smaller product J2R recombined with the neoR gene, which was introduced as a selection marker during construction of VVΔEΔK+RhTRS1 (Fig. S3C). These two sites represented the predominant recombination sites (85.5% and 2.8% respectively) identified by Illumina deep sequencing. However, we found additional minor recombination sites by Illumina deep sequencing, including a 15 kb duplication in VV-B. The presence of an identical recombination site in all three passaged virus pools suggests that duplication may have been present at a very low frequency in the initial virus population even though we did not detect it in the Illumina sequencing data. Regardless, taken together these data demonstrate that the copy number of rhtrs1 in the viral genome was substantially increased by passage through PRO1190 cells.

Unlike a previous study which identified adaptive point mutations arising after locus expansion [17], we did not detect any point or indel mutations in rhtrs1 in any of the passaged virus pools. However, we identified 13 vaccinia virus gene mutations present at greater than 5% frequency in at least one of the pools (Table 1). All three passaged pools had one or more of four different single nucleotide deletions within the A35R gene at frequencies ranging from 12 to 42%. Transition mutations affecting the A24R and A37R genes were present at >50% frequencies in VV-A and VV-B respectively, but were rare or absent in the other viral pools. None of the other mutations were detected in all three pools or occurred at >50% frequency in any pool, so are unlikely to account for the improved replication of the passaged viruses. However, the presence of these VV gene mutations raised the question of whether the expanded species tropism we observed was due to the VV gene mutations or to rhtrs1 amplification.

If rhtrs1 amplification alone is sufficient for the observed increase in fitness, we reasoned that overexpression of rhtrs1 in trans might rescue VVΔEΔK+RhTRS1 replication. To investigate this possibility, we stably transduced rhtrs1 into HFF (HFF+RhTRS1), and confirmed RhTRS1 expression by immunoblot (data not shown). We also prepared a control cell line (HFF-LHCX) by transducing the empty vector, LHCX, into HFF. In the control cells, VVΔEΔK+RhTRS1 replicated approximately 1000-fold less efficiently than VV-βg (Fig. 5A). In HFF+RhTRS1, VVΔEΔK+RhTRS1 replication increased more than 100-fold. Thus, combined expression of RhTRS1 from genes in both the cell and the infecting virus potentiated VVΔEΔK+RhTRS1 replication, supporting the hypothesis that rhtrs1 amplification alone is sufficient to expand the species tropism of VVΔEΔK+RhTRS1.

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larger image TIFF original image Download: Figure 5. RhTRS1 overexpression or PKR knockdown permits efficient VVΔEΔK+RhTRS1 replication in HFF. (A) Viral production 48 hpi of control (HFF+LHCX, grey bars) or HFF expressing RhTRS1 (HFF+RhTRS1, black bars) with the indicated viruses (MOI = 0.1), determined by titering on HFF+TRS1. (B) Viral production 48 hpi of control (HFF-ctrl kd) or HFF with PKR knocked down by shRNA (HFF-PKR kd) with the indicated viruses (MOI = 0.1), determined by titering on BSC40 cells. Data are represented as mean +1 STD. https://doi.org/10.1371/journal.ppat.1004002.g005

Although rhtrs1 amplification provided a substantial growth benefit in HFF and RF, the passaged viruses still replicated 100- to 1000-fold less efficiently than VV-βg (Fig. 3). To determine whether this incomplete rescue in HFF was due to incomplete PKR inhibition or represented a second block to replication, we infected HFF stably transduced with either a PKR specific shRNA (HFF-PKR kd) that reduces PKR expression >95%, or a non-specific shRNA (HFF-ctrl kd) [22]. Knocking down PKR increased VVΔEΔK+RhTRS1 replication ∼1000-fold, indicating that PKR is a major barrier to replication in these cells (Fig. 5B). All three passaged virus pools replicated ∼10-fold better in HFF-PKR kd cells than in HFF-ctrl kd cells, suggesting that rhtrs1 amplification, which fully inhibits PRO1190 PKR, only partially inhibits human PKR. However, these viruses all replicated ∼10-fold less well than VV-βg in the HFF-PKR kd cells. This remaining replication defect may be due to incomplete PKR knockdown in these cells [20], although it is also possible that an additional host factor inhibits VVΔEΔK+RhTRS1 replication in HFF.