Polymycovirus Infection Sensitizes Aspergillus fumigatus for Antifungal Effects of Nikkomycin Z

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94–1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.


Introduction
Virus infections are widespread in the fungal kingdom, and since the 1970s, many Aspergillus wild-type strains have been naturally infected [1]. Aspects of virus infection of Aspergillus, including effects on fungal physiology and virulence, have been recently reviewed and detailed [1,2]. Among the viruses infecting Aspergillus is the Polymycoviridae family, which was initially reported in 2015 [3], and received an official ICTV virus taxonomy profile in 2022 [4]. The Polymycoviridae family currently accommodates one genus, Polymycovirus, with 10 species, including Aspergillus fumigatus polymycovirus. Polymycoviridae and related viruses usually have four to eleven [5,6] dsRNA genomic segments. The majority of polymycoviruses are not conventionally encapsidated [3,5], although filamentous particles have been reported in one case [7], and are infectious as dsRNA [3,7,8]. Little is known about how viral infection affects Aspergillus physiology or virulence. Where effects on fungal physiology have been delineated, the results have shown either enhancing or deleterious effects [1,2].
Af293 is a very common and fully sequenced A. fumigatus laboratory strain [9], naturally infected with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) [3]. Using naturally  [3] The naturally infected Af293 strains 10-53 and 18-95 were kept in separate laboratories (10-53 in the US, 18-95 in the UK) for over 10 years. Strains 18-95 were cured from AfuPmV-1 using the protein synthesis inhibitor cycloheximide [3], resulting in strains 18-42, 18-42B, and 18-42C. While strains 18-42, 18-42B, and 18-42C were different strains derived similarly from the same curing process of 18-95. Strains 18-95B and 18-95C are separate clones of 18-95, each verified as containing the virus. Purified AfuPmV-1 was re-introduced in the virus-free Aspergillus 18-42 by protoplast transfection [3], producing the reinfected strain used here, . The presence or absence of AfuPmV-1 was confirmed by Northern blotting and RT-qPCR as previously described [3]. ments ( Figure 1A). Microscopic evaluation showed that NikZ, at the concentrations tested, interfered with fungal growth as well, but did not prevent hyphal formation completely, even at the highest concentration used, 15 µg/mL ( Figure 1B). NikZ caused the formation of balloon-like or knob-like structures in hyphae that were not observed in untreated controls ( Figure 1B). These structures, at a lesser size and frequency, occurred at NikZ concentrations that had no significant effects on fungal metabolism, i.e., NikZ 0.06 µg/mL (compare Figure 1A to Figure 1B).

NikZ Dose-Dependently Interfered with Forming Biofilm Metabolism of Two A. fumigatus Reference Strains and Caused Structural Changes in Hyphae
NikZ significantly interfered with the metabolism of forming biofilms of 10AF at concentrations > 0.47 µg/mL, with the IC50 between 0.94 and 1.88 µg/mL in most experiments ( Figure 1A). Microscopic evaluation showed that NikZ, at the concentrations tested, interfered with fungal growth as well, but did not prevent hyphal formation completely, even at the highest concentration used, 15 µg/mL ( Figure 1B). NikZ caused the formation of balloon-like or knob-like structures in hyphae that were not observed in untreated controls ( Figure 1B). These structures, at a lesser size and frequency, occurred at NikZ concentrations that had no significant effects on fungal metabolism, i.e., NikZ 0.06 µg/mL (compare Figure 1A to Figure 1B).  When NikZ (2 µg/mL) effects were compared between the virus-free strain A. fumigatus strain 10AF and the naturally AfuPmV-1-infected A. fumigatus strain Af293 (10-53), we found 10-53 to be more sensitive to NikZ than 10AF (Figure 2A). Microscopic evaluation showed that, at the NikZ concentration used in Figure 2A, balloon-like structures occurred in both A. fumigatus strains ( Figure 2B); the frequency of these structures also appears dose-related. The IC50 for NikZ against infected Af293 strains 10-53, 18-95, and 19-40 was determined to be between 0.94 and 1.88 µg/mL ( Figure 2C). µg/mL) at 37 °C for 16 h. Aspergillus metabolism was measured by XTT assay. Metabolism in the presence of RPMI alone (white bar) was regarded as 100%. The red line represents the IC50. Comparisons: No NikZ (white bar) vs. each NikZ concentration (grey bars). Statistical analysis (n = 6): Unpaired t-test, three asterisks = p ≤ 0.001. Only significant differences are indicated. (B) Pictures were taken from wells described in A after 16 h of incubation at 37 °C before XTT analysis. A representative picture is shown. All pictures were taken at 125× magnification.

Infected Af293 Is More Sensitive to NikZ Than Virus-Free Af293
We next compared three Af293-based strains for their sensitivity to NikZ, of which two strains (18-95 and 19-40) were infected, and a third strain, 18-42, was virus-free (see Section 2 for details). Our results show that both infected strains were significantly more sensitive to NikZ than the virus-free strain ( Figure 3A). A dose-response curve for NikZ on 18-42 revealed the IC50 to be between 3.8 and 7.5 ug/mL ( Figure 3B), which is 2 to 4 times higher than the IC50 on infected Af293 strains (compare Figure 3B to Figure 2C). In addition to 18-42, we used two more cured, virus-free isolates, 18-42B and C, which, like 18-42, were obtained separately by curing 18-95 from AfuPmV-1 infection. These virus-free isolates, like 18-42, were significantly more resistant to NikZ than 18-95, with an IC50 ≥ 5 µg/mL ( Figure 3C).

Infected Af293 Is More Sensitive to NikZ Than Virus-Free Af293
We next compared three Af293-based strains for their sensitivity to NikZ, of which two strains (18-95 and 19-40) were infected, and a third strain, 18-42, was virus-free (see Section 2 for details). Our results show that both infected strains were significantly more sensitive to NikZ than the virus-free strain ( Figure 3A). A dose-response curve for NikZ on 18-42 revealed the IC50 to be between 3.8 and 7.5 ug/mL ( Figure 3B), which is 2 to 4 times higher than the IC50 on infected Af293 strains (compare Figure 3B to Figure 2C). In addition to 18-42, we used two more cured, virus-free isolates, 18-42B and C, which, like 18-42, were obtained separately by curing 18-95 from AfuPmV-1 infection. These virusfree isolates, like 18-42, were significantly more resistant to NikZ than 18-95, with an IC50 ≥ 5 µg/mL ( Figure 3C). (white bar) was regarded as 100%. The red line represents the IC50. Comparisons: No NikZ (white bar) vs. each NikZ concentration (grey bars). Statistical analysis (n = 4): Unpaired t-test, two and three asterisks = p ≤ 0.01 and p ≤ 0.001, respectively. Only significant differences are indicated. Concentrations 0.94 or 0.06 µg/mL also did not demonstrate inhibition and are not shown here to simplify presentation. (C) Infected (18-95B, 18-95C) or virus-free (18-42B, 18-42C) Aspergillusforming biofilm was incubated with NikZ (0.31 or 5 µg/mL) at 37 • C for 16 h. Fungal metabolism was measured by XTT assay. Metabolism in the presence of RPMI alone (leftmost bars) was regarded as 100%. Comparisons: No NikZ vs. all NikZ concentrations for each strain. Other comparisons as indicated by the ends of the brackets. Statistical analysis (n = 8): 1-way ANOVA, one, two, or three asterisks = p ≤ 0.05, p ≤ 0.01, or p ≤ 0.001, respectively. Only significant differences are indicated.

AfuPmV-1 Infection Does Not Sensitize A. fumigatus Biofilm to Conventional Drugs
In assays similar to those used to determine the NikZ effects shown in Figure 3A Figure 5B). These results for conventional drugs were similar to those obtained for planktonic fungal growth in a recent study by our group [10].   or virus-free (18-42) Aspergillus-forming biofilm was incubated with NikZ (2 or 4 µg/mL) at 37 °C for 16 h. Fungal metabolism was measured by XTT assay. Metabolism in the presence of RPMI alone (white bars for each strain) was regarded as 100%. Comparisons: No NikZ (white bars) vs. NikZ (black and grey bars) for each strain. Other comparisons are indicated by the ends of the brackets. Statistical analysis (n = 4): Unpaired t-test, three asterisks = p ≤ 0.001. Only significant differences are indicated. (B) Strains 18-42 forming biofilm were incubated with NikZ (15 to 0.06 µg/mL) at 37 °C for 16 h. Fungal metabolism was measured by XTT assay. Metabolism in the presence of RPMI alone (white bar) was regarded as 100%. The red line represents the IC50. Comparisons: No NikZ (white bar) vs. each NikZ concentration (grey bars). Statistical analysis (n = 4): Unpaired t-test, two and three asterisks = p ≤ 0.01 and p ≤ 0.001, respectively. Only significant differences are indicated. Concentrations 0.94 or 0.06 µg/mL also did not demonstrate inhibition and are not shown here to simplify presentation. (C) Infected (18-95B, 18-95C) or virus-free (18-42B, 18-42C) Aspergillus-forming biofilm was incubated with NikZ (0.31 or 5 µg/mL) at 37 °C for 16 h. Fungal metabolism was measured by XTT assay. Metabolism in the presence of RPMI alone (leftmost bars) was regarded as 100%. Comparisons: No NikZ vs. all NikZ concentrations for each strain. Other comparisons as indicated by the ends of the brackets. Statistical analysis (n = 8): 1-way ANOVA, one, two, or three asterisks = p ≤ 0.05, p ≤ 0.01, or p ≤ 0.001, respectively. Only significant differences are indicated.

AfuPmV-1 Infection Does Not Sensitize A. fumigatus Biofilm to Conventional Drugs
In assays similar to those used to determine the NikZ effects shown in Figure 3A, we examined the effects of four drugs conventionally used for the treatment of A. fumigatus infections. Virus infection of A. fumigatus did not increase its sensitivity to MICA ( Figure  4A), CASPO ( Figure 4B), VCZ ( Figure 5A), or AmB ( Figure 5B). These results for conventional drugs were similar to those obtained for planktonic fungal growth in a recent study by our group [10].

Virus-Free Planktonic Af293 Is Less Sensitive to NikZ Than Infected Fungus
We now were interested in how NikZ affected AfuPmV-1-infected compared to virus-free Af293. Our results showed that NikZ effects on virus-free Af293 (strain 18-42) were weaker than on infected strains ( Table 2). With respect to the inhibition of 50% of growth (MIC 50%), we found a two-fold difference between infected and virus-free strains. With respect to 95% inhibition of growth (MIC 95%, only traces of fungus visible), the difference was at least 3-to 4-fold. Total inhibition of visible growth (MIC 100%), as well as sterilization of the inoculum (MFC) for all strains, was higher than the highest tested NikZ concentration of 128 µg/mL ( Table 2).

Virus-Free Planktonic Af293 Is Less Sensitive to NikZ Than Infected Fungus
We now were interested in how NikZ affected AfuPmV-1-infected compared to virusfree Af293. Our results showed that NikZ effects on virus-free Af293 (strain 18-42) were weaker than on infected strains ( Table 2). With respect to the inhibition of 50% of growth (MIC 50%), we found a two-fold difference between infected and virus-free strains. With respect to 95% inhibition of growth (MIC 95%, only traces of fungus visible), the difference was at least 3-to 4-fold. Total inhibition of visible growth (MIC 100%), as well as sterilization of the inoculum (MFC) for all strains, was higher than the highest tested NikZ concentration of 128 µg/mL (Table 2).

Increased Growth or Metabolism Does Not Explain Decreased Sensitivity of Virus-Free Aspergillus to NikZ
We observed that virus-free Af293, growing planktonically ( Figure 6A), as well as in the form of biofilm ( Figure 6B), grew significantly better than infected Af293.

Increased Growth or Metabolism Does Not Explain Decreased Sensitivity of Virus-Free Aspergillus to NikZ
We observed that virus-free Af293, growing planktonically ( Figure 6A), as well as in the form of biofilm ( Figure 6B), grew significantly better than infected Af293. Similarly, biofilm assays showed that virus-free fungus had significantly more metabolism than infected fungus ( Figure 7A). We considered whether its increased ability to grow rendered virus-free fungus less sensitive to NikZ.
We measured biofilm metabolism in 18-42 cultures that started with the same amount of conidia as used in Figure 7A, 2500 conidia per well, diluted stepwise to as little as 1/8 of that concentration, 313 conidia per well ( Figure 7B). At increasing starting conidial concentrations, increased levels of metabolic activity were measured at the end of the assay. Comparing metabolism between these conidia concentrations for 18-42 to metabolism of 19-40 (2500 conidia/well), we found similar metabolism between 19-40 (2500 conidia/well) and 18-42 (625 conidia/well) ( Figure 7B). When NikZ (2 µg/mL) was assessed with these two conidial concentrations, and metabolism of the control for each conidial concentration was defined as 100%, it became clear that even when normalized to equal metabolism (625 conidia/well in 18-42; 2500 in 19-40), virus-free fungus was still more resistant to NikZ ( Figure 7C). Similarly, biofilm assays showed that virus-free fungus had significantly more metabolism than infected fungus ( Figure 7A). We considered whether its increased ability to grow rendered virus-free fungus less sensitive to NikZ.
We measured biofilm metabolism in 18-42 cultures that started with the same amount of conidia as used in Figure 7A, 2500 conidia per well, diluted stepwise to as little as 1/8 of that concentration, 313 conidia per well ( Figure 7B). At increasing starting conidial concentrations, increased levels of metabolic activity were measured at the end of the assay. Comparing metabolism between these conidia concentrations for 18-42 to metabolism of 19-40 (2500 conidia/well), we found similar metabolism between 19-40 (2500 conidia/well) and 18-42 (625 conidia/well) ( Figure 7B). When NikZ (2 µg/mL) was assessed with these two conidial concentrations, and metabolism of the control for each conidial concentration was defined as 100%, it became clear that even when normalized to equal metabolism (625 conidia/well in 18-42; 2500 in 19-40), virus-free fungus was still more resistant to NikZ ( Figure 7C).
This study showed that even when we compensated for the differences in basal metabolism between virus-free and infected, NikZ effects on infected strains were still greater. Thus, the increased basal metabolism of the virus-free strain does not explain the decreased NikZ effect on it.

Discussion
Virus infection of Aspergillus is not a rare event. There are multiple virus families with members that are known to infect Aspergillus, encompassing Chrysoviridae, Narnaviridae, Partitiviridae, Totiviridae, and Polymycoviridae [20]. The infection rate by mycoviruses among Aspergillus species has been described to be between 10 and 80% [20,21], while between 7 and 19% of clinical A. fumigatus isolates are infected [22,23]. Infection with mycoviruses seems to be latent, persistent, and difficult to eliminate [20,24].
The clinical relevance of Aspergillus virus infection is controversial. There are reports showing hypovirulence in a mouse model [25], increased virulence [1,26,27], or no change in virulence whether infected or not infected [1,27,28]. In two studies using planktonic fungal growth, no altered susceptibility towards conventional antifungal drugs has been detected [10,28].
We also found that virus-free and mycovirus-infected A. fumigatus strains, in the form of planktonically growing cultures, but also in the form of biofilms, were comparably sensitive to conventional drugs affecting fungal membranes (voriconazole, amphotericin B), or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms as well as planktonic cultures of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and between 0.94 and 1.88 µg/mL for infected isogenic Af293 strains. The IC50 for the virus-free A. fumigatus strain 10AF was approximately 2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of AfuPmV-1-infected Af293 more than it affected the virus-free strain (MIC 50% virus-free 4 µg/mL, infected 2 µg/mL). Virus-free Af293 biofilm showed increased metabolism, and when growing in the form of biofilm, or planktonically, showed increased growth compared to infected Af293.
Our results indicate that virus infection of Aspergillus renders the fungus more susceptible to stresses affecting cell wall formation, especially towards NikZ, affecting chitin synthase. It would be of interest to compare the effects of other chitin synthase inhibitors, e.g., [29], to NikZ effects on infected vs. virus-free fungus in future studies. Stress induced by other inhibitors of cell wall synthesis (echinocandins) is not amplified by viral infection, indicating specificity for the kind of cell wall stress.
We observed that virus-free fungus grew better compared to infected fungus, suggesting that viral infection might impair cell wall synthesis, particularly at specific points in the synthesis of the wall. Although we could show that resistance of virus-free biofilm to NikZ was not caused by its better growth, we indicate that virus-altered cell wall synthesis is more prone to detrimental effects caused by inhibition of chitin synthesis. This inhibition might result in osmotic stress for the fungal cell, resulting from impaired cell wall synthesis, and either lead to reduced growth or cell injury.
In contrast to our data, infection of a plant pathogenic fungus, Penicillium digitatum, with a polymycovirus resulted in increased susceptibility to an azole drug [8]. Although Penicillium and Aspergillus are both filamentous fungi, the higher susceptibility of infected Penicillium to azole drugs might be explained by differences in cell wall stress response [30].
We previously showed that virus infection of A. fumigatus alters responses to a variety of stresses, such as iron deficiency [10,11], Pseudomonas volatiles [10], or high salt [13]. Whether virus infection renders A. fumigatus more susceptible to other stresses is currently under investigation. Higher susceptibility of infected strains to a variety of stresses points to the virus affecting molecules that have been proposed as master regulators of stress responses or cell wall stress responses [31][32][33]. It might also be possible that under stress conditions, virus accumulation would increase and further increase host sensitivity, e.g., to NikZ. Further studies need to investigate these hypotheses.