ABSTRACT
The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear, as recombinant viruses containing a single mutation have not been analyzed. We obtained AMNV-resistant viruses with amino acid substitutions by several passages under AMNV treatment. Twenty HSV-1 and 19 HSV-2 mutants with mutation(s) in UL5 helicase and/or UL52 primase, but not in cofactor UL8, were isolated. The mutations in UL5 were located downstream of motif IV, with UL5 K356N in HSV-1 and K355N in HSV-2, in particular, identified as having the highest frequency, which was 9/20 and 9/19, respectively. We generated recombinant AMNV-resistant HSV-1 with a single amino acid substitution using bacterial artificial chromosome (BAC) mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants and showed equivalent viral growth in vitro and virulence in vivo as the parent HSV-1, although other mutants showed attenuated virulence. All recombinant viruses were susceptible to the other antiherpetic drugs, acyclovir and foscarnet. In conclusion, based on BAC mutagenesis, this study identified, for the first time, mutations in UL5 and UL52 that contributed to AMNV resistance and found that a mutant with the most frequent K356N mutation in HSV-1 maintained viral growth and virulence equivalent to the parent virus.
KEYWORDS: HSV-1, HSV-2, antiviral agents, amenamevir, helicase-primase complex, UL5, UL52
INTRODUCTION
Herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) are widely prevalent pathogens belonging to the family Herpesviridae. HSVs and VZV establish lifelong latent infection in sensory ganglia after primary infection and subsequently reactivate, leading to recurrent episodes. HSV infections are one of the most widespread infectious diseases in the world, with 60 to 95% of the population infected with at least one of these viruses (1). Three classes of drugs that target viral DNA replication are approved for the treatment of HSV infections, acyclic guanosine analogues, acyclic nucleotide analogues, and pyrophosphate analogues (2). Typical drugs from these three classes include acyclovir (ACV) and penciclovir and their prodrugs valaciclovir and famciclovir, respectively, and these have become the gold standard for the treatment and prophylaxis of HSV and VZV infections (3–8). However, due to active replication of the viruses in immunocompromised patients and the long-term use of these antiherpetic drugs, drug-resistant viral strains have developed (2).
ACV, one of the nucleotide analogues, is selectively phosphorylated to a monophosphate derivative in infected cells by virus-encoded thymidine kinase (TK). As the affinity of ACV for HSV-TK is more than 200-fold higher than that for human TK, ACV shows low toxicity in humans (2). Cellular kinases convert the monophosphate derivatives of ACV to diphosphate- and triphosphate (TP)-active derivatives. ACV-TP inhibits not only viral polymerase function but also DNA replication by competitively replacing dGTP and, therefore, the efficacy of ACV depends on the ratio of ACV-TP to dGTP (3, 9).
On the other hand, foscarnet (FOS), one of the pyrophosphate analogues, inhibits the function of viral polymerases without activation or phosphorylation of the compounds by viral or cellular proteins. FOS inhibits the function of herpesvirus DNA polymerase through the exchange of pyrophosphate from deoxynucleoside triphosphate during viral replication by directly binding to a site on the viral DNA polymerase (10, 11).
HSVs and VZV cause diseases in immunocompromised hosts and are a major cause of their morbidity and mortality. Prolonged antiviral treatment is often required for the clinical management of herpesvirus infection in immunocompromised patients, and this can lead to the development of drug-resistant viral strains (2, 12). The isolation of ACV-resistant HSV-1, HSV-2, and VZV strains has been reported with increasing frequency (12–14). FOS is recommended for use in the treatment of ACV-resistant virus infection (15–17); however, double resistance to both ACV and FOS has been observed in immunocompromised patients after sequential and concomitant treatment with either or both drugs (12, 14, 18). For the diagnosis of drug-resistant HSVs and VZV, a genotyping test is frequently performed (19). Although ultradeep sequencing or real-time cell analysis has improved our ability to detect variants and mixed populations of drug-resistant viruses (20, 21), it is difficult to diagnose and detect HSV- and VZV-resistant mutants at present in a clinical setting. The emergence of such potential multiresistant mutants represents a growing concern. Therefore, novel antiherpetic drugs, apart from nucleotide and pyrophosphate analogues, with alternative mechanisms of action that are highly effective and exhibit low toxicity against ACV- or FOS- resistant HSVs are desired.
The helicase-primase complex performs essential functions in viral DNA replication and viral growth (22). This complex consists of three proteins, helicase, primase, and cofactor subunits, which are well conserved among viruses belonging to the family Herpesviridae. The genes encoding the HSV helicase (UL5), primase (UL52), and cofactor (UL8) subunits share homology with the open reading frame 55 (ORF55), ORF 6, and ORF52 genes of VZV, respectively (23, 24). The helicase-primase complex possesses multienzymatic activities, including a single-stranded DNA-dependent ATPase, a 5′ to 3′ helicase, and primase activities. All of these enzymatic activities are needed for the helicase-primase complex to function in viral DNA replication in vivo, although the UL8 protein was found to not necessarily be required for helicase and primase activities in some assays (25). Therefore, agents that target the helicase-primase complex have been seen as potential drugs for the treatment of herpesviruses. The amino-thiazolylphenyl-containing compound, BILS 179 BS, and pritelivir (BAY 57 to 1293), a thiazole urea derivative, have been developed as helicase-primase inhibitors (HPIs) with anti-HSV activity (26, 27). Amenamevir (AMNV) is an HPI with an oxadiazolephenyl moiety that possesses antiviral activity against not only HSV-1 and HSV-2 but also VZV (24, 28). AMNV received authorization for commercial use throughout Japan for the first time in any country in 2017 and has been used to treat herpes zoster, which is caused by the reactivation of latent VZV (29). HSV-1 and HSV-2 are also susceptible to AMNV, as all 50% effective concentration (EC50) values for HSV-1 and HSV-2 of BILS 179 BS, pritelivir, and AMNV are between 0.01 to 0.06 μM (24). Therefore, the indications of AMNV for use against genital herpes, herpes labialis, and herpes keratitis are expected to be expanded (30).
Characterization of each antiherpetic drug-resistant mutant would be beneficial to the selection of the most appropriate antiherpetic drug. HPI-resistant mutants have been reported in vitro (27, 31–33). Mutants resistant to HSV-specific HPIs, BILS 179 BS, and pritelivir were previously characterized in terms of resistance, replication in vitro, and pathogenicity in mice (31). AMNV-resistant mutants of not only HSV but also VZV were also investigated (24, 34). However, these studies analyzed mutated viruses that were obtained by AMNV selection and did not examine whether the mutations confer AMNV resistance to HSV by reconstitution of the mutation.
Here, we obtained AMNV-resistant HSVs carrying mutations that were either newly identified in this study or have been reported elsewhere. Furthermore, we generated recombinant AMNV-resistant HSV mutants containing a single amino acid substitution. We also analyzed the replication and resistance to AMNV, ACV, and FOS in vitro in addition to the pathogenicity in vivo of these recombinant viruses (Fig. 1). As a result, we were able to identify the mutations critical to AMNV resistance.
FIG 1.
Workflow of this study and the strategy of generating the bacmids for the mutant viruses. (A) Workflow of each step is shown. (1) Selection of AMNV-resistant viruses. (2) Analyzing the sensitivity to each drug and the UL5 or UL52 sequences of the selected viruses, followed by generation of the recombinant viruses. (3) Analyzing the characteristics and pathogenicity of the recombinant viruses. (B) DNA fragment consisting of the kanamycin-resistant gene flanked by sequences homologous to the wild-type genome is amplified using the indicated primer pair. Each segment (a, b, c, and d) contains 15 to 20 bases homologous to the UL5 or UL52 gene, and the target base(s) is flanked by segment b and c. Black circles, target base(s); crosses, substitution base(s).
RESULTS
Isolation of AMNV-resistant HSV-1 and HSV-2 mutants.
To identify HSV mutations with AMNV resistance, we infected Vero cells with HSV-1 or HSV-2 in the presence of AMNV and obtained AMNV-resistant mutants by plaque-purified isolation. We then conducted a plaque reduction assay to compare the AMNV susceptibility of the selected mutants and their parent viruses (Fig. 1). Twenty clones of HSV-1 and 19 clones of HSV-2 were recognized as AMNV-resistant clones due to their elevated EC50 values for AMNV compared to those of their parent viruses (Table 1). Clones showing an EC50 value for AMNV higher than 0.15 μM or 3-fold higher than the wild type were determined to be AMNV resistant. As AMNV-resistant viruses are reported to possess mutations in the coding region of the helicase-primase complex, we investigated the sequences of three genes, UL5, UL8, and UL52, in the mutant genomes. DNA sequence analyses identified several single-base-pair substitutions resulting in amino acid changes in the UL5 helicase and UL52 primase but not in the UL8 cofactor of the AMNV-resistant mutants in comparison to their parent viruses (Table 1). We found a number of novel and previously reported AMNV-resistant mutations (34). Motif IV of UL5 is known to be responsible for HSV DNA replication. Most mutations endowing resistance to HPIs have been observed slightly downstream of this motif (32, 34–36). The AMNV-resistant viruses in our study also possessed mutations in this region, and some of them (K356N of HSV-1 and K355N and G351R of HSV-2) showed double-digit higher resistance to AMNV than did the other mutants. Interestingly, additional mutations in the UL52 genes, Y222C of HSV-1 (the R4 strain) and F363S of HSV-2 (the r13 strain), restored the susceptibility of K356N of HSV-1 and K355N of HSV-2 by about 80- and 10-fold, respectively.
TABLE 1.
Amino acid substitutions in UL5 or UL52 of AMNV-resistant HSV-1 or HSV-2 clonesa
| Strain | Mutation site |
AMNV EC50 (μM) | |
|---|---|---|---|
| UL5 | UL52 | ||
| HSV-1 | |||
| Wild type | 0.06 ± 0.01 | ||
| R18 | K356N | 194.4 ± 12.98b | |
| R19 | K356N | 187.6 ± 6.89b | |
| R1 | K356N | 182.4 ± 8.33b | |
| R8 | K356N | 171.7 ± 15.50b | |
| R3 | K356N | 166.7 ± 18.02b | |
| R13 | K356N, E34K | 166.0 ± 16.05b | |
| R9 | K356N | 153.3 ± 21.52b | |
| R12 | K356N, S341I | 143.7 ± 32.77b | |
| R4 | K356N | Y222C | 2.12 ± 0.21b |
| R11 | M355T | 2.20 ± 0.22b | |
| R17 | M355T | F294L | 0.63 ± 0.14 |
| R20 | M355T | 0.55 ± 0.11b | |
| R6 | M355T | 0.26 ± 0.07 | |
| R2 | G352V, G477D | 2.00 ± 0.04b | |
| R15 | G352C | 1.66 ± 0.45 | |
| R5 | G352C | 0.70 ± 0.06* | |
| R23 | F360V | 8.43 ± 1.04b | |
| R10 | F360C | 1.75 ± 1.09 | |
| R7 | S364G N902T | 1.98 ± 0.21b | |
| R21 | N902T | 0.92 ± 0.35 | |
| HSV-2 | |||
| Wild type | 0.05 ± 0.01 | ||
| r7 | K355N | 184.9 ± 7.17b | |
| r9 | K355N | 184.6 ± 5.79b | |
| r14 | K355N | 167.6 ± 12.61b | |
| r8 | K355N | 156.9 ± 11.96b | |
| r2 | K355N | 134.6 ± 15.41b | |
| r11 | K355N | 124.1 ± 2.84b | |
| r3 | K355N | 113.8 ± 4.78b | |
| r10 | K355N | 112.2 ± 11.17b | |
| r13 | K355N | F363S | 16.73 ± 0.93b |
| r1 | G351R | 109.8 ± 36.46 | |
| K355E | 6.45 ± 0.75b | ||
| r21 | K355R | T31A | 1.24 ± 0.41 |
| r20 | K355R | 0.41 ± 0.15 | |
| r16 | K355R | 0.40 ± 0.16 | |
| r6 | K355R | 0.40 ± 0.12 | |
| r12 | M354I | S397G | 2.16 ± 0.39b |
| r4 | I340V | 0.16 ± 0.03 | |
| r15 | N97T | S290N | 0.93 ± 0.16b |
| r19 | T39A | H913Q | 0.35 ± 0.16 |
Amino acid substitutions in UL5 or UL52 of AMNV-resistant HSV-1 and HSV-2 clones, respectively, are shown. The mean EC50 values ± standard error (SE) of each clone to AMNV were calculated from duplicate measurements. Clones showing an EC50 value for AMNV higher than 0.15 μM were determined to be AMNV resistant.
P < 0.05. Data are based on three independent experiments.
Susceptibility of AMNV-resistant mutants to ACV and FOS.
To examine whether AMNV-resistant mutants are also resistant to ACV or FOS, we conducted a plaque reduction assay of the AMNV-resistant mutants for ACV and FOS. None of the HSV-1 or HSV-2 mutants showed higher EC50 values to ACV (HSV-1, 1.04 to 6.64 μM; HSV-2, 2.62 to 8.30 μM) and FOS (HSV-1, 27.5 to 379 μM; HSV-2, 34.5 to 210 μM) than did their respective parental viruses (HSV-1, 5.62 ± 1.13 μM, and HSV-2, 3.42 ± 0.66 μM for ACV; HSV-1, 288 ± 94.8 μM, and HSV-2, 157 ± 56.6 μM for FOS). These results revealed that none of the HSV-1 or HSV-2 AMNV-resistant mutants was cross-resistant to ACV or FOS.
Frequency of the substitution of amino acid residues in AMNV-resistant viruses.
The structure of helicase is conserved among Herpesviridae and contains six well-conserved motifs. In 16 of the 20 AMNV-resistant HSV-1 mutants, amino acid substitutions existed downstream of the motif IV, with K356N being the most frequently observed in 9 of 20 mutants (Fig. 2 and Table 1). In the AMNV-resistant HSV-2 mutants, 17 of the 19 mutants possessed mutations around motif IV, with the substitutions of K355, which corresponds to K356 of HSV-1, being particularly common (observed in 14 of the 19 mutants).
FIG 2.
Emergent frequencies of the mutations around motif IV of UL5 in AMNV-resistant HSV-1 and HSV-2. The amino acid sequence near motif IV of UL5 of HSV-1 and HSV-2 and the mutated amino acid residues of AMNV-resistant clones along with the emergent frequencies are shown. The rectangle surrounds the amino acid sequence corresponding to motif IV.
In the HSV-1 mutants, some mutations in the UL52 primase were clearly related to AMNV resistance.
Generating recombinant AMNV-resistant HSV-1.
To confirm whether the identified mutations contribute to AMNV resistance, we reconstituted mutations in the UL5 or UL52 of HSV-1 by bacterial artificial chromosome (BAC) mutagenesis (Fig. 1; Table 2). Each recombinant strain contained one amino acid substitution that had been identified, as shown in Table 1. The revertant UL5 mutants were restored for four substitutions (K356N, G352C, G352V, and M355T) simultaneously. We then conducted a plaque reduction assay to compare the susceptibility to AMNV of the respective recombinant strains of HSV-1 and their parent virus named bacF. We found that four recombinant clones possessing K356N, G352C, G352V, or M355T of UL5 and three clones carrying F360V, F360C, or N902T of UL52 contributed to AMNV resistance based on the fact that the EC50 values of these clones were elevated in comparison with the parental virus. Moreover, we confirmed the wild-type drug susceptibility of each revertant of the AMNV-resistant recombinant clones.
TABLE 2.
AMNV sensitivity of recombinant HSV-1 containing a single amino acid residue substitutiona
| Strain | Mutation site | AMNV EC50 (μM) | |
|---|---|---|---|
| Parent recombinant virus | 0.02 ± 0.00 | ||
| UL5 | K356N | 5.22 ± 0.85b | |
| G352C | 0.50 ± 0.04c | ||
| G352V | 0.06 ± 0.13c | ||
| M355T | 0.12 ± 0.01b | ||
| E34K | 0.03 ± 0.01c | ||
| S341I | 0.03 ± 0.01c | ||
| G477D | 0.03 ± 0.01c | ||
| UL52 | F360V | 3.17 ± 0.32 | |
| F360C | 0.50 ± 0.13c | ||
| N902T | 0.18 ± 0.00b | ||
| Y222C | 0.03 ± 0.02c | ||
| F294L | 0.03 ± 0.01c | ||
| S364G | 0.02 ± 0.01c | ||
| Revertant virus | UL5 | 352-356 | 0.03 ± 0.01c |
| UL52 | F360V/C | 0.02 ± 0.00c | |
| N902T | 0.03 ± 0.01c | ||
Recombinant strains of HSV-1 with a single amino acid residue substitution and the respective EC50 values are shown. Revertant virus 352 to 356 in which mutations such as K356N, G352C, G352V, and M355T in UL5 were restored simultaneously. The mean EC50 values ± SE of each clone to AMNV were calculated from duplicate measurements. Clones showing an EC50 value 3-fold higher than the wild type were determined to be AMNV resistant. Data are based on three independent experiments. The novel mutations contributing to AMNV resistance, G352C of UL5 and F360C, F360V, and N902T of UL52, are underlined.
P < 0.05.
Not significant.
On the other hand, recombinant clones possessing E34K, S341I, or G477D mutations in UL5 and Y222C, F294L, or S364G mutations in UL52 showed equivalent EC50 values to the parent virus. The Y222C mutation in UL52 had no effect on susceptibility to AMNV but did have an effect on counteracting the resistance induced by K356N in UL5, as observed in the R4 strain (Table 1).
Susceptibility of recombinant AMNV-resistant viruses to ACV and FOS.
We also conducted a plaque reduction assay to compare the ACV and FOS susceptibility of the recombinant AMNV-resistant HSV-1 strains and the parent virus. The EC50 values for ACV and FOS of recombinants were 0.53 to 3.04 μM and 13.0 to 39.8, respectively, and there were no differences from those for the parent (4.56 ± 1.08 μM for ACV and 37.7 ± 3.83 μM for FOS). These results suggested that none of the recombinant AMNV-resistant strains of HSV-1 had cross-resistance to ACV or FOS.
Growth of recombinant AMNV-resistant HSV-1 viruses.
The growth rates were compared between recombinant AMNV-resistant mutants and the parent virus with a one-step growth experiment (Fig. 3). Mutants carrying G352V or M355T in UL5 or F360V in UL52 showed significantly lower replication capacity than the parent (bacF), although the other recombinant viruses replicated to the same degree as the parent.
FIG 3.
Replication of AMNV-resistant recombinant virus clones in a one-step growth experiment. PFU per milliliter for each AMNV-resistant HSV-1 recombinant clone, the parent virus, and the revertant viruses are shown. Error bars show means ± SE. *, P < 0.05. Data are representative of at least three independent experiments.
Pathogenicity of recombinant AMNV-resistant HSV-1.
In order to examine whether the virulence of AMNV-resistant strains differs from that of their parent in vivo, mice were infected intracranially with each recombinant AMNV-resistant strain, the revertant strain, or the parent virus. The survival curves for each mouse group inoculated with UL5 mutants (Fig. 4A) or with UL52 mutants are shown (Fig. 4B). The virulence of the recombinant virus with K356N in UL5 was not significantly different from that of the parent virus (P = 0.202). Although the recombinant virus with M355T showed relatively lower pathogenicity, viruses possessing G352C or G352V had significantly lower pathogenicity than did the parent. Furthermore, all mice inoculated with the virus possessing G352V survived throughout the observation period. The recombinant viruses carrying F360V/C or N902T in UL52 were significantly attenuated compared with both the parent and revertant strains. From these results, it was suggested that K356N of UL5 was the source of the pathogenicity of HSV, although most AMNV-resistant mutations seem to attenuate virulence in vivo.
FIG 4.
Survival of mice infected with AMNV-resistant recombinant HSV-1 clones. (A) Kaplan-Meier curves of the mouse groups inoculated intracranially with 10 PFU/mouse of the UL5-mutated recombinant virus, the parent, and the revertant virus are shown, respectively. (B) Kaplan-Meier curves of the mouse groups inoculated with 103 PFU/mouse of the UL52-mutated recombinant virus, the parent, and the revertant viruses are shown, respectively. The statistical difference between each recombinant virus-inoculated group and the parent-inoculated group was determined by the log-rank test. A P value of <0.05 was regarded as statistically significant.
Comparison of amino acid sequences among HSV-1 strains.
The whole-genome sequences of 41 HSV-1 strains, which are thought not to have been previously exposed to AMNV, were obtained from GenBank, and the sequences of the UL5 and UL52 genes were compared with those of the VR-3 strain (Fig. 5). The amino acid residues in HSV-1, which were confirmed to endow AMNV resistance, are shown. The substitutions in the AMNV-resistant mutants identified in this study or previous reports were not found in the sequences of the viruses obtained from GenBank, except for S364 in UL52. These results suggested that the viruses possessing AMNV-resistant mutations seldom arise spontaneously. Moreover, Y222C, which could restore the susceptibility of the K356N mutation, was not observed as a polymorphism in the 41 strains containing the F strain.
FIG 5.
Comparison of amino acids in the helicase UL5 and primase UL52 among HSV-1 strains in GenBank. The positions of amino acid residues that differ between the helicase UL5 and the primase UL52 of HSV-1 (VR-3) are shown as each slit (upper row, clinical isolates and laboratory strains obtained from GenBank; middle row, mutations in AMNV-resistant viruses in this study; lower row, HPI-resistant mutations in previous studies). Scheme of the helicase sequence with the conserved motifs (gray box) is displayed.
DISCUSSION
In this study, a number of single-base-pair substitutions that have the potential to confer AMNV resistance in HSVs were found in the helicase- or primase-coding region of the genome of AMNV-resistant HSV-1 and HSV-2. Subsequently, using recombinant HSV-1 viruses generated by BAC mutagenesis, we identified a number of single amino acid substitutions that are responsible for AMNV resistance.
We identified the mutation of three amino acid residues, G352, M355, and K356, in the UL5 gene of HSV-1 that are responsible for AMNV resistance. Moreover, the replacement of G352 with C was detected as a novel substitution. These results are compatible with those of previous reports that the resistance to other HPIs, pritelivir, and BILS 179 BS is conferred by the mutation of one of four amino acid residues, G352, M355, K356, or N342 (27, 32, 33, 35). Chono et al. also reported that amino acid mutations of G352 and M355 were identified in AMNV-resistant HSV-1 (34). These amino acid residues are located immediately downstream of motif IV of UL5 (32, 34, 35). Motif IV is one of the most conserved domains among the six helicase motifs of Herpesviridae viruses and is required to support HSV DNA replication by ATPase activity (25, 36). The HPIs directly inhibit the ATPase activity of the helicase-primase complex (26, 27, 32, 35). As previously reported, an α-helical region of UL5, formed by F351 to K356, appears to be the center of virus interaction with HPIs, such as BILS 179 BS and pritelivir (35), and this region is immediately downstream of motif IV. The mutation sites in AMNV-resistant HSV-1 identified in this study, i.e., G352, M355T, and K356N, as well as G351, M354, and K355 in HSV-2, are also located in this region. Our results also showed that the importance of the C-terminal amino acid of the region being a basic amino acid, as the mutation of K355 to R in HSV-2 had a markedly weaker effect on AMNV sensitivity in comparison to a substitution to N.
Furthermore, the growth of some AMNV-resistant clones of HSV-1 possessing mutations around motif IV, such as G352C, M355T, and K356N in combination with S341I, were strongly impaired, although the clones bearing the K356N mutation alone replicated to the same degree as the parent virus. These results were similar to those of previous reports that showed the growth of HSV demonstrating resistance to other HPIs with mutations except for K356N is impaired (32, 35, 37). Moreover, Biswas et al. mentioned that the three aromatic rings of pritelivir were involved in the interaction with UL5 (35). As AMNV also contains three aromatic rings, AMNV may also interact with UL5 at the α-helical region and block the ATPase function of the helicase-primase complex of HSV in a manner similar to that in pritelivir.
In this study, we revealed, for the first time, that the N902T mutation in UL52 is involved in the AMNV resistance conferred by a single amino acid substitution. Previous reports showed that A897T or A899T mutations in UL52 confer HSV-1 resistance to HPI in collaboration with an M355V or K356 mutation in UL5, as the A899 of UL52 shows spatial proximity to the regions in the UL5 subunit that are expected to interact with pritelivir (27, 35, 38). Therefore, the N902 near A899 in UL52 may also be involved in the interaction of AMNV with UL5 so that the mutation of N902 may result in AMNV resistance.
We also found that a single mutation F360V/C in UL52 is involved in AMNV resistance. Although the S364G mutation in UL52 was also reported to be detected within AMNV-resistant HSV-1 (34), our findings did not show this mutation to contribute to AMNV resistance via a single amino acid substitution. We also revealed that the AMNV-resistant HSV-1 strain containing both S364G and N902T mutations in UL52 showed 2-fold higher AMNV resistance than the strain containing the N902T mutation alone. The amino acid residues 367 to 421 in UL52 are thought to interact strongly with the helicase UL5 subunit (39); therefore, F360V/C and S364G mutations existing near the amino acid residues may play an important role in enhancing AMNV resistance alone or in combination with other mutations in UL52, such as N902T.
Most AMNV-resistant HSV-1 mutants containing the K356N mutation in UL5 showed more than 2,000-fold higher EC50 values than did the parent virus. On the other hand, the R4 strain, which contains Y222C in UL52 in addition to K356N, showed only 35-fold higher EC50 values than did the parent. These results suggested that a specific mutation, such as Y222C in UL52, could attenuate the AMNV resistance resulting from K356N in UL5. Structural analysis may help to reveal the function of Y222 in the binding of AMNV to the helicase-primase complex.
We showed that recombinant HSV-1 carrying a K356N or G352C mutation in UL5, as well as F360C or N902T in UL52, showed equivalent growth to that of the parent. On the other hand, the mutants possessing G352V or M355T in UL5 and F360V in UL52 exhibited lower replication than did the parent. In addition, the recombinant HSV-1 possessing K356N showed similar pathogenicity to the parent strain, although the virulence of the other recombinant viruses was reduced in vivo. Previous studies reported that HPI-resistant HSV-1 containing G352V/R or both G352V and M355I in UL5 showed equivalent or reduced growth in vitro and decreased virulence in vivo compared to the wild-type virus (33–35, 37). However, viruses with K356Q or K356N in UL5 were reported to show similar or increased viral growth and equal virulence compared to their parent (27, 32). Therefore, the substitution of K356 confers AMNV resistance on HSV without any loss of replication ability or pathogenicity in vivo. Thus, careful analysis of clinical cases is required for the identification of emerging resistant viruses.
The K356N mutants among the AMNV-resistant mutants, R1, R3, R8, R9, R18, and R19, showed extremely high resistance to AMNV (Table 1). However, the recombinant strains possessing the same mutation showed relatively lower EC50 values (Table 2). There is a possibility that this is due to the differences in the genetic background between the parent VR-3 and bacF strains such as Y222C in UL52 of the R4 strain.
In conclusion, by generating recombinant HSV-1 strains using BAC mutagenesis, we identified novel mutations in UL5 and UL52 that confer AMNV resistance. There is a possibility that AMNV-resistant HSVs may emerge with high frequency, particularly virulent viruses with K356 mutations in UL5. Alternative AMNV treatments for ACV-resistant virus infections or multidrug interventions using AMNV and ACV could be developed, as there is no cross-resistance to these reagents (40). Further structural analysis is needed to clarify the interaction between the helicase-primase complex and AMNV.
MATERIALS AND METHODS
Antiviral drugs.
AMNV and ACV were provided by Maruho and GlaxoSmithKline, respectively, and foscarnet (FOS) was purchased from Thermo Fisher Scientific (catalog no. P6801).
Cell and viruses.
Vero cells were grown at 37°C in 5% CO2 with Dulbecco’s modified Eagle’s medium (Nissui Biosciences, Tokyo, Japan) supplemented with 10% heat-inactivated newborn cow serum (NCS; Thermo Fisher Scientific), glutamine (0.292 g/liter), penicillin (50 U/ml), streptomycin (250 μg/ml), and amphotericin B (1.25 μg/ml) (DMEM-NCS10). The recombinant HSV-1 (strain F) (kindly provided by Y. Kawaguchi, The University of Tokyo) (41), HSV-1 strain VR-3, and HSV-2 strain UW-268 were propagated using Vero cells in DMEM-NCS2 and stored at −80°C until use. The VR-3 and UW-268 strains are antiherpetic drug-sensitive laboratory strains (42, 43).
Selection of AMNV-resistant HSV-1 and HSV-2 mutants.
AMNV-resistant HSV strains were isolated by serial passage of the reference VR-3 and UW-268 strains in the presence of increasing concentrations of AMNV as follows (Fig. 1). Each virus (200 PFU/well) was inoculated onto Vero cell monolayers in a 24-well tissue culture plate, and the infected cultures were incubated in a medium containing 0.01 μM AMNV. After 2 to 3 days, 1:500 of the supernatant of each well was transferred to new wells in which noninfected Vero cells were cultured. AMNV concentrations were stepwise increased from 0.01 μM to 0.5 μM during 18 serial passages of each supernatant. After a final passage in 0.5 μM AMNV, each virus population emerging from the independent cultures was serially diluted, inoculated onto Vero cell monolayers in a 24-well culture plate, and cultivated in DMEM-NCS2 supplemented with 0.8% (wt/vol) methylcellulose no. 4000 (Nacalai Tesque, Kyoto, Japan). Every single plaque was selected from each independent virus population using a micropipette under microscopy and was reinoculated onto fresh Vero cells. After two cycles of plaque purification, the virus suspensions were obtained and stored at −80°C as cell-free AMNV-resistant HSV-1 and HSV-2 mutants.
Plaque reduction assay.
Vero cells monolayered confluently in 24-well cell culture plates were infected with each HSV-1 or HSV-2 strain at approximately 60 PFU/well in DMEM-NCS2. The medium was removed after incubation for 1 h at 37°C, and then 1 ml of a medium containing 0.8% methylcellulose number 4000 and either AMNV, ACV, or FOS was added. Each drug was prepared with 6 graduated concentrations starting with 0 μM to the maximum concentration (AMNV, 200 μM; ACV, 10 μM; and FOS, 400 μM). After plaques appeared, the cells were fixed with 10% formalin in phosphatase-buffered saline (Nissui Biosciences, Tokyo, Japan) and stained with 0.05% crystal violet solution (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan). The number of plaques in each well was counted, and the EC50 values for the HSV-1 or HSV-2 infected cells were then calculated for each drug.
Sequencing analyses.
Viral genomic DNA was extracted from Vero cells infected with each HSV-1 or HSV-2 strain. The BAC genome was purified using a Qiagen Plasmid Plus Midi kit (Qiagen, Hilden, Germany). DNA regions, including UL5 (helicase), UL52 (primase), and UL8, were amplified via PCR using a proofreading DNA polymerase (Kod FX, Toyobo, Japan) and the corresponding primer sets (HSV-1 UL5, forward, 5′-tgaacctttacccagccgtcct-3′, and reverse, 5′-tgtggattggacatctcgcggt-3′; UL52, forward, 5′-gcgctccccagccacatatag-3′, and reverse, 5′-aatacatcggtgcagcggact-3′; and UL8, forward, 5′-aggcacggcagcatgggacccacaga-3′, and reverse, 5′-gtattcggacagagactcaagca-3′) (HSV2 UL5, forward, 5′-gatgtaggctgtacgcgatggt-3′, and reverse, 5′-tgaaccttcacccagccgtcct-3′; UL52, forward, 5′-aacacgcgcgcggctctgcgc-3′, and reverse, 5′-ggactggcaccaaagacgatgta-3′; and UL8, forward, 5′-tataagtctggggccgcgctcgttc-3′, and reverse, 5′-gccagtaaatacggacgcgcgcac-3′). PCR amplicons were used for direct sequencing with a BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Waltham, MA, US). Sequences of all genomic DNAs were confirmed by Sanger sequencing (catalog no. ABI3130xl; Thermo Fisher Scientific, Waltham, MA, US) performed on both DNA strands in order to rule out any PCR artifacts, followed by analysis with GENETYX software (version 8.1.0; Genetyx, Tokyo, Japan).
One-step growth experiment.
Vero cells were adsorbed with each HSV strain or the mutant at a multiplicity of infection (MOI) of 1 at 37°C for 24 h, followed by the storage of each infected culture at −80°C. The cell-free viruses were obtained after three freeze-thaw cycles followed by centrifugation at 800 × g for 5 min at 4°C to remove cell debris. The virus titer in each sample was determined by plaque assay on Vero cells. Briefly, Vero cells monolayered confluently in 24-well cell culture plates were infected with serially 10-fold diluted sample virus suspensions for 1 h at 37°C. After discarding the virus suspension, 1 ml of DMEM-NCS2 containing 0.8% methylcellulose number 4000 (Nacalai Teaque, Inc., Kyoto, Japan) was overlaid on the infected Vero cells. The cultures were incubated for 4 days in a CO2 incubator, and the number of plaques was observed.
BAC mutagenesis.
The recombinant viruses were generated using Escherichia coli GS1783 containing the BAC of the HSV-1 (strain F) genome (pYEbac102) (kindly provided by Y. Kawaguchi of The University of Tokyo) as described elsewhere (Fig. 5B; 43–46). Briefly, the DNA fragment for homologous recombination to generate amino acid residue substitutions in UL5 or UL52, and revertants were amplified using the pEP-KanS plasmid as a template (kindly provided by N. Osterrieder, Cornell University) and the primers listed in Table 3, yielding amplicons of approximately 1.1 kb (Fig. 1B). The fragment was introduced into pYEbac102-containing GS1783 by electroporation (Gene Pulser II, Bio-Rad Laboratories, Hercules, CA), and fragment-transfected E. coli was then incubated at 30°C for 2 days in the presence of kanamycin (20 μg/ml) and chloramphenicol (10 μg/ml). The kanamycin-resistant sequence was removed by treatment with 2% arabinose to generate E. coli containing pYEbac102 and possessing the mutation. Successful recombination of the mutants and the revertants was confirmed by PCR and Sanger DNA sequencing of the whole target gene. The bacmids for the revertants were constructed by transduction of the DNA fragment into GS1783 bearing the corresponding mutant BAC. The purified bacmids were transfected into Vero cells to reconstitute an infectious recombinant virus. The revertant viruses were confirmed by comparing phenotypes such as drug resistance (EC50 value), one-step growth, and pathogenicity with the parent virus.
TABLE 3.
Primer sets for generating recombinant HSV-1 viruses that have a point mutation and for the respective revertant viruses
| Strain | Mutation site | Primer | |
|---|---|---|---|
| Mutant | UL5 | K365N | Forward, 5′-cgagcacgagttcggtaacctcatgaatgtgctggagtacggcctgcccatcaccgaggatgacgacgataagta-3′ |
| Reverse, 5′-ctgcatgtgctcctcggtgatgggcaggccgtactccagcacattcatgaggttcaaccaattaaccaattctgattag-3′ | |||
| G352C | Forward, 5′-aacaacaaacggtgcgtcgagcacgagttctgtaacctcatgaaggtgctggagtacgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-cggtgatgggcaggccgtactccagcaccttcatgaggttacagaactcgtgctccaaccaattaaccaattctgattag-3′ | |||
| G352V | Forward, 5′-ccatttttattaacaacaaacggtgcgtcgagcacgagttcgttaacctcatgaaggtaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-aggccgtactccagcaccttcatgaggttaacgaactcgtgctcgacgcaccgttcaaccaattaaccaattctgattag-3′ | |||
| M355T | Forward, 5′-ccatttttattaacaacaaacggtgcgtcgagcacgagttcggtaacctcacgaaggtaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-aggccgtactccagcaccttcgtgaggttaccgaactcgtgctcgacgcaccgttcaaccaattaaccaattctgattag-3′ | |||
| E34K | Forward, 5′-accgaccagccgttccagggagggccaaggcctttttaaattttacgtctatgcacggaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-ggattggctgcaccccgtgcatagacgtaaaatttaaaaaggccttggccctcccaaccaattaaccaattctgattag-3′ | |||
| S314I | Forward, 5′-accagaaactgcggtgttccgtccgccagatcgagaacgtgctcacgtacctcatctgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-cgcagcgtgcggttgcagatgaggtacgtgagcacgttctcgatctggcggacggcaaccaattaaccaattctgattag-3′ | |||
| G477D | Forward, 5′-cgaataccgacggctgacacaccagcccgacctgactattgaaaagtggctcacggccaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-gatgcggctggcgttggccgtgagccacttttcaatagtcaggtcgggctggtgtcaaccaattaaccaattctgattag-3′ | |||
| UL52 | F360V | Forward, 5′-accgtgaattcattacgtacatctacctggcccatgttgagtgtttcagccccccgcgaggatgacgacgataagtaggg-3′ | |
| Reverse, 5′-agatgcgtggctaggcgcggggggctgaaacactcaacatgggccaggtagatgtcaaccaattaaccaattctgattag-3′ | |||
| F360C | Forward, 5′-accgtgaattcattacgtacatctacctggcccattgtgagtgtttcagccccccgcgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-agatgcgtggctaggcgcggggggctgaaacactcacaatgggccaggtagatgtcaaccaattaaccaattctgattag-3′ | |||
| N902T | Forward, 5′-atgtgagcttctttgaaaggaaggcgtcccgcaccgcgctggaacactttgggcgacgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-tccgtcagggtctcgcgtcgcccaaagtgttccagcgcggtgcgggacgccttcccaaccaattaaccaattctgattag-3′ | |||
| S364G | Forward, 5′-cattacgtacatctacctggcccattttgagtgtttcggccccccgcgcctagccacgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-cacggcccgaagatgcgtggctaggcgcggggggccgaaacactcaaaatgggcccaaccaattaaccaattvtgattag-3′ | |||
| V222C | Forward, 5′-gcgcgtgcttgccgcgtaccgcagggcgtattgtggaagcgcgcagagtcccttctggaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-gaatttgctaagaaaccagaagggactctgcgcgcttccacaatacgccctgcggcaaccaattaaccaattctgattag-3′ | |||
| F294L | Forward, 5′-ccccccgccccgacaccgtcagcgctgcgtccctgacctcgcttgccgccatcacgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-cgtgcaacagaaccgcgtgatggcggcaagcgaggtcagggacgcagcgctgacgcaaccaattaaccaattctgattag-3′ | |||
| Revertant | UL5 | 352-356 | Forward, 5′-aacaacaaacggtgcgtcgagcacgagttcggtaacctcatgaaggtgctggagtacgaggatgacgacgataagtaggg-3′ |
| Reverse, 5′-cggtgatgggcaggccgtactccagcaccttcatgaggttaccgaactcgtgctccaaccaattaaccaattctgattag-3′ | |||
| UL52 | F360V/C | Forward, 5′-accgtgaattcattacgtacatctacctggcccattttgagtgtttcagccccccgcgaggatgacgacgataagtaggg-3′ | |
| Reverse, 5′-agatgcgtggctaggcgcggggggctgaaacactcaaaatgggccaggtagatgtcaaccaattaaccaattctgattag-3′ | |||
| N902T | Forward, 5′-atgtgagcttctttgaaaggaaggcgtcccgcaacgcgctggaacactttgggcgacgaggatgacgacgataagtaggg-3′ | ||
| Reverse, 5′-tccgtcagggtctcgcgtcgcccaaagtgttccagcgcgttgcgggacgccttcccaaccaattaaccaattctgattag-3′ | |||
In vivo virulence analysis of AMNV-resistant recombinant HSV-1.
Female BALB/c mice at 3 weeks of age were purchased from CLEA Japan (Tokyo, Japan). Four or five mice per group were injected intracranially under anesthesia with 5 μl of one of the 10-fold serially diluted virus suspensions. Survival of each mouse group was observed daily for 14 days, and then survival curves were constructed.
Statistical analysis.
To compare the EC50 values, statistical differences were determined by the Student's t test for the parent HSV and the AMNV-resistant or -revertant strains. Kaplan-Meier survival analysis and log-lank test were performed to compare survival rates among mouse groups using SPSS software version 25 (IBM Corporation, Armonk, New York, US). A P value of <0.05 was considered statistically significant.
DNA and amino acid sequence data.
We obtained DNA sequences from NCBI GenBank. Accession numbers of the analyzed genomic DNA sequences of HSV-1 are as follows: GU734771.1 (strain F), MH999851.1, MH999850.1, MH999849.1, MH999848.1, MH999847.1, MH999846.1, MH999845.1, MH999844.1, MH999842.1, MH999841.1, MH999840.1, MH999839.1, MH102298.1, KY922719.1, KY922718.1, MN136523.1, JQ780693.1, KF498959.1, MN159383.1, MN159379.1, MN159378.1, MN159377.1, MN136524.1, NC_001806.2, MF156584.1, KX424525.1, GU734772.1, AB618031.1, LT594457.1, LT594192.1, LT594112.1, LT594111.1, LT594110.1, LT594109.1, LT594107.1, LT594106.1, and LT594105.1. The DNA sequences of the HSV-1 VR-3 strain UL5, UL8, and UL52 genes and the HSV-2 UW-268 strain UL5, UL8, and UL52 genes have been submitted to the DDBJ database and have been assigned accession nos. LC613247 to LC613252, respectively. The sequences of the mutated UL5 or UL52 of each clone of the AMNV-resistant HSV-1 VR-3 strain in Table 1 were assigned the DDBJ accession nos. LC635546 (R1, R3, R4, R8, R9, R18, and R19), LC635547 (R13), LC635548 (R12), LC635549 (R6, R11, R17, and R20), LC635550 (R2), LC635551 (R5 and R15), LC635552 (R4), LC635553 (R17), LC635554 (R23), LC635555 (R10), LC635556 (R7), and LC635557 (R21). The DDBJ accession nos. LC636205 (r2, r3, r7, r8, r9, r10, r11, r13, and r14), LC636206 (r1), LC636207 (r23), LC636208 (r6, r16, r20, and r21), LC636209 (r12), LC636210 (r4), LC636211 (r15), LC636212 (r19), LC636213 (r13), LC636214 (r21), LC636215 (r12), LC636216 (r15), and LC636217 (r19) correspond to the mutated UL5 or UL52 sequences of the HSV-2 UW-268 strain of each clone in Table 1.
ACKNOWLEDGMENTS
We thank K. Nishiyama, R. Kanno, and C. Ozaki for their technical assistance. We also thank T. Koshizuka for his thoughtful comments and Maruho Co. Ltd. for generously providing the amenamevir.
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