Divergent pathogenic dynamics of immature tick-borne and mosquito-borne flaviviruses: a paradigm shift in prM-containing particle infectivity

doi:10.21203/rs.3.rs-3918672/v1

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Divergent pathogenic dynamics of immature tick-borne and mosquito-borne flaviviruses: a paradigm shift in prM-containing particle infectivity

https://doi.org/10.21203/rs.3.rs-3918672/v1

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Maturing flaviviruses undergo pH-dependent conformational changes, transitioning from trimeric pre-membrane protein prM and envelope protein E (prM-E) complexes to dimeric E with cleaved prM. However, little is known about the intermediate maturation stages and whether immature virions contribute to infection. Here we demonstrated that immature prM-containing particles of tick-borne flaviviruses—tick-borne encephalitis virus (TBEV), Langat virus, and louping ill virus—exhibited infectivity in mammalian cells under physiological conditions. This finding challenges the long-standing assumption that prM-containing flavivirus particles are non-infectious, as is the case for immature mosquito-borne flaviviruses, including Usutu virus, Zika virus, and West Nile virus (WNV). In a mouse model, prM-TBEV exhibited high pathogenicity, contrasting with the reduced pathogenicity of prM-WNV. Structural modelling, functional validation, and site-directed mutagenesis revealed interactions within the prM-E complex that influence pH sensitivity, furin cleavage site accessibility, and virus infectivity. This study, therefore, elucidates the distinctive dynamics of infectivity of tick- and mosquito-borne flaviviruses.

Biological sciences/Microbiology/Virology/Virus–host interactions

Biological sciences/Microbiology/Virology/Viral pathogenesis

Flaviviruses—genus Orthoflavivirus (formerly genus Flavivirus), family Flaviviridae—are responsible for millions of human infections and numerous fatalities annually¹. They constitute a diverse group of arboviruses transmitted by tick or mosquito vectors, which cause a spectrum of clinical syndromes in humans, including life-threatening encephalitides, haemorrhagic fevers, and birth defects². Notable members include tick-borne encephalitis virus (TBEV; Orthoflavivirus encephalitidis)³, West Nile virus (WNV; Orthoflavivirus nilense)⁴, Japanese encephalitis virus (JEV; Orthoflavivirus japonicum)⁵, Zika virus (ZIKV; Orthoflavivirus zikaense)⁶, and dengue virus (DENV; Orthoflavivirus denguei)⁷.

Mosquito-borne flaviviruses (MBFV) most commonly cause human infections, but tick-borne flaviviruses (TBFV) also constitute an emerging group that significantly impacts people across expansive geographic regions⁸. For instance, TBEV causes over 10,000 cases annually of tick-borne encephalitis (TBE) across Eurasia, predominantly affecting central, eastern, and northern Europe, and northeast Asia⁹. Recent detections of TBEV in the British Isles¹⁰ and North Africa¹¹ indicate a broader geographic range for this virus.

Flaviviruses are small, enveloped viruses carrying an approximately 11-kilobase-long positive-sense single-stranded RNA genome (+ ssRNA), including a single open reading frame that encodes a large polyprotein. This polyprotein sequentially undergoes co-translational and post-translational modifications facilitated by viral and cellular proteases¹², which result in the generation of seven non-structural and three structural proteins: capsid protein (C), pre-membrane protein (prM), and envelope protein (E)¹³.

For TBEV and other flaviviruses, the life cycle starts with binding to the host cell receptor, after which the virus is internalized through clathrin-mediated endocytosis. Within the endosome, the acidic pH triggers conformational changes in envelope protein E, inducing a fusion state. Subsequent fusion of the viral envelope with the endosomal membrane facilitates viral RNA release into the host cell cytoplasm^{14, 15}.

Flavivirus replication complexes are situated at the endoplasmic reticulum (ER) membrane. Here, newly synthesized structural proteins (E, prM, and C) undergo budding into the ER, forming nascent viral particles that simultaneously acquire lipid envelopes^{16, 17}. The prM protein comprises an N-terminal globular "pr" domain, and an extended "M" domain that anchors the entire protein within the membrane¹⁸.

Within the neutral pH environment of the ER, virions assemble into immature particles organized as prM-E trimers, forming spike-like structures. These immature particles traverse the secretory pathway through the Golgi and trans-Golgi network, encountering an acidic milieu in the Golgi complex. The low pH triggers structural rearrangement of the spiky virions into smooth, yet still immature, particles containing prM-E dimers, which are oriented tangentially to the cell membrane¹⁹.

These structural alterations expose the cleavage site on the prM protein, enabling the cellular furin protease to cleave the "pr" portion of prM. Additionally, the pH-induced E protein rearrangement marks the final stages of viral maturation, crucial for infectivity^{20, 21}. The prM protein functions as a molecular chaperone, preventing pH-dependent conformational changes associated with membrane fusion, blocking the fusion loop. Consequently, the "pr" component remains associated with the cleaved virus particle until it is released into the extracellular environment, whereupon it dissociates due to the neutral pH^{21, 22, 23, 24}.

Furin cleavage efficacy can vary²⁵, sometimes releasing partially or completely immature particles. Partially mature viruses retain infectivity, while immature prM-containing particles are considered non-infectious²¹. For MBFV, the prM-E conformational changes between neutral and acidic pH are reversible. In contrast, for TBEV, the pH-induced prM-E conformational changes are irreversible, such that the distinction between neutral and acidic pH is critical in TBEV maturation^{20, 26}. Recently, the structure of spiky immature TBEV particles was determined, confirming these observations²⁷.

Here we performed a comprehensive series of in vitro and in vivo experiments and demonstrated that immature prM-containing particles of TBEV, produced in furin-deficient cells, exhibited full infectivity. This trait was also exhibited by other TBFV, including Langat virus (LGTV; Orthoflavivirus langatense) and louping ill virus (LIV; Orthoflavivirus loupingi). In contrast, consistent with previous findings^{28, 29}, prM-containing particles from MBFV—including Usutu virus (USUV; Orthoflavivirus usutuense), WNV, and ZIKV—displayed non-infectivity or significantly reduced infectivity. This newly-identified divergence between tick-borne and mosquito-borne flaviviruses profoundly influences the pathogenic potential of TBEV.

Furthermore, our investigations utilizing in vitro TBFV-/MBFV-derived peptide cleavage assays revealed consistent processivity of furin protease over a broad pH range. Through bioinformatic structural modelling and site-directed mutagenesis, we identified specific residues that are critical for regulating interactions within the prM-E complex. Importantly, these residues significantly impact the accessibility of the prM cleavage site to furin, in response to pH changes and prM-E complex stability.

Our findings provide novel and substantial insights into flavivirus maturation mechanisms, elucidating distinct differences in infectivity between tick-borne and mosquito-borne variants.

Production and characterization of prM-TBEV

By infecting furin-deficient human LoVo cells, we produced immature prM-containing TBEV virions³⁰ (prM-TBEV) (Fig. 1a). These prM-TBEV and mature TBEV (m-TBEV, derived from BHK-21) were analysed by western blot. The m-TBEV sample was mostly cleaved, with a thin prM band indicating a low abundance of immature particles. In contrast, the prM-TBEV sample contained mostly uncleaved prM protein, indicating predominantly immature virions (Fig. 1b).

We quantified prM-TBEV in the supernatant using RT-qPCR and a plaque assay (Fig. 1c). Both analyses yielded similar titres: ~10⁵ PFU ml^− 1 in the plaque assay, and ~ 10⁶ viral copies per ml by RT-qPCR. Surprisingly, this suggested that the prM-TBEV virions might be infectious.

We next tested prM-TBEV virus infection in three mammalian cell lines (PS, BHK-21, and Vero) and the vector tick cell line IRE/CTVM19. After infection at MOI = 0.1, viral infectivity was determined by plaque assay at 48 hours post-infection (hpi) (mammalian cell lines) and 7 days post-infection (dpi) (IRE/CTVM19) (Fig. 1d). These results showed that prM-TBEV had infectivity similar to that of m-TBEV in all cell lines tested.

Furin-mediated cleavage during virus entry

Furin cleavage is described mainly during virus maturation in Golgi apparatus (GA), but furin is also found in endosomes and on the cell surface³¹. To assess furin utilization during prM-TBEV entry, we infected LoVo cells with m-TBEV and prM-TBEV virus (MOI 1). Virus samples were harvested at 48 hpi, and the titres differed between groups by 4 log₁₀ (P < 0.0001) (Fig. 1e).

Next, TBEV virus producing fluorescent mCherry protein was propagated in LoVo cells to obtain a prM-containing reporter variant (prM-mCherry-TBEV). LoVo cells infected with mCherry-TBEV exhibited a red fluorescent signal, which was absent in cells infected with prM-mCherry-TBEV (Fig. 1f). This suggested that the reduced prM-TBEV and prM-mCherry-TBEV infection was likely due to the absence of furin. We further used the furin inhibitor Decanoyl-RVKR-CMK to ablate furin cleavage in a plaque reduction assay. Confluent PS cells were pretreated for 2 h with furin inhibitor (0, 10, 50, and 100 µM) and then infected with mCherry-TBEV and prM-mCherry-TBEV. Significant inhibition of prM-mCherry-TBEV by almost 80% was observed, especially at 50 and 100 µM. In comparison, mCherry-TBEV was inhibited by only 35% (P < 0.01) (Fig. 1g, h). Together, these results confirmed the necessity of furin cleavage for maturation and demonstrated that this final maturation step can be efficiently performed during entry.

Immature prM-flavivirus infectivity in TBFV and MBFV

We further investigated whether prM-containing immature particles of other TBFV and MBFV were infectious, as shown for prM-TBEV. Using LoVo cells, we produced prM samples for other flaviviruses transmitted by ticks (LGTV, LIV) and mosquitoes (USUV, WNV, ZIKV). Next, we performed a plaque assay with each prM flavivirus sample, to determine the numbers of infectious virions. All tick-borne prM variants showed high infectivity for mammalian cells, with titres of ~ 10⁴ PFU ml^− 1 (prM-LGTV), ~10⁵ PFU ml^− 1 (prM-TBEV), and ~ 10⁷ PFU ml^− 1 (prM- LIV). In contrast, MBFV prM showed low infectivity (prM-WNV ~ 10² PFU ml^− 1) or no infectivity (prM-ZIKV and prM-USUV) (P < 0.0001) (Fig. 2a).

To confirm that MBFV could productively replicate in LoVo cells and produce immature non-infectious virions, we performed RT-qPCR and calculated the ratio of viral RNA copies to infectious plaque-forming units for immature and mature USUV and WNV (Fig. 2b, c), and TBEV (Fig. 2d). These results showed that the specific infectivities of prM-USUV and prM-WNV were at least 1,000-fold and 10,000-fold lower than those of m-USUV (P < 0.01) and m-WNV (P < 0.01), respectively. In contrast, the specific infectivities of prM-TBEV and m-TBEV differed by less than 10-fold (P < 0.01). Although TBFV and MBFV are closely related, their immature prM virions showed dramatically different infectivities, which could have further implications regarding their overall biology.

pH-dependent furin cleavage in TBEV and USUV

To assess the pH dependence of furin cleavage, we performed in vitro cleavage experiments using recombinant human furin (r-furin). prM-TBEV infection of LoVo cells was examined at neutral (7.5) and acidic (5.5) pH, with or without r-furin. Under both pH conditions, LoVo cells were infected in the presence of r-furin (titres > 10⁵ PFU ml^− 1), but not in its absence (P < 0.01) (Fig. 2e). In addition, pH dependence was investigated using a western blot, which confirmed that furin cleavage is efficient throughout the pH range tested (Supplementary Fig. 2), implying that furin cleavage can occur during the entry steps. We next tested the infection of PS cells with prM-USUV, which occurred only at acidic pH, with or without r-furin (P < 0.01). This indicated that endogenous furin from host cells could be utilized during prM-USUV infection at acidic pH (Fig. 2f,g). In comparison, prM-TBEV infected PS cells at both pH levels, with or without r-furin (Fig. 2g). Together, these data showed that maturation cleavage of prM-TBEV virions occurs independently of pH, whereas pH has important effects on maturation cleavage of prM-USUV. This highlights different pH-dependent dynamics of structural proteins, with irreversible or reversible structural changes observed for TBFV and MBFV, respectively (Fig. 2h).

Pathogenicity of prM-TBEV and prM-WNV in a mouse model

In a mouse model, we examined the pathogenicity of prM-TBEV and prM-WNV, together with m-TBEV and m-WNV. For each virus variant, we established three groups of female BALB/c mice, which were inoculated subcutaneously (s.c.) with different doses: 10¹, 10², or 10³ viral genomic equivalents (gen. eq.). For 28 days, survival rates and clinical signs were monitored and scored. Doses of 10² and 10³ gen. eq. of m-TBEV or m-WNV showed lethality rates of ~ 90%, as previously determined (Fig. 3 and Supplementary Fig. 3a, b). The highest doses of mTBEV and prM-TBEV caused fatal infections in all mice between days 8 and 12 (Supplementary Fig. 3a). Among mice infected with 10² gen. eq., one mouse survived infection with m-TBEV (Fig. 3a). With the lowest infectious dose, 60% of m-TBEV-infected mice survived, compared to 40% of prM-TBEV-infected mice (Supplementary Fig. 3a). Overall, prM-TBEV and m-TBEV showed similar pathogenicity and reached similar viral titres in sera and brains collected 3 and 8 dpi, respectively (Fig. 3d, e).

In experiments with m-WNV, 10³ and 10² gen. eq. resulted in lethal infection, whereas we observed 60% survival following infection with 10¹ gen. eq. (Fig. 3f-h, Supplementary Fig. 3b). Interestingly, all mice survived following infection with 10¹ and 10² gen. eq. of prM-WNV (P < 0.0001), without signs of neuroinfection, confirmed by plaque assay from the sera and brains (Fig. 3i,j). Only 20% of mice survived following infection with 10³ gen. eq. of prM-WNV (Supplementary Fig. 3b). Using calculations based on a ratio of PFU ml^− 1 to viral RNA copies per ml and with an infection dose of 10³ gen. eq., inoculation of prM-WNV contained approximately 7 infectious virions per mouse. This caused fatal disease in 80% of mice, with a course of infection similar to that in mice infected with 10¹ gen. eq. of m-WNV (~ 10 PFU/mouse). Overall, prM-WNV exhibited dramatically reduced infectivity in vivo.

Structural prM-E interface and its effect on furin cleavage site accessibility

Furin cleavage and pH dependence could be influenced by different amino acid compositions of the furin cleavage site and neighbouring regions, altering the binding efficiency of the furin protease to the furin cleavage site. The prM and E proteins of tick-borne and mosquito-borne flaviviruses share ~ 30–40% sequence homology (Fig. S3, S4) and involve intricate interactions at the prM cleavage site and E protein interface. We hypothesized that several ionic interactions in and around the furin cleavage site critically regulate its steric accessibility. To investigate this, we designed four internally quenched fluorogenic substrates based on prM sequences of two TBFV (TBEV, LGTV) and two MBFV (WNV, USUV), conjugated to Edans (donor for the FRET assay) and the quencher Dabcyl (Fig. 4a). For each substrate, Km values (Michaelis constant) were measured in fluorescence assays, showing that MBFV-derived peptides were good substrates for r-furin (Km values: 1.6 ± 0.33 µM for WNV, and 2.1 ± 0.35 µM for USUV) (Supplementary Fig. 4). In contrast, the TBFV-derived peptides were poor substrates (Km values: 4.3 ± 0.68 µM for TBEV, and 5.0 ± 0.55 µM for LGTV) (Supplementary Fig. 4). We next investigated the pH profile (4.0–9.5) of each substrate around its Km value. Interestingly, all tested substrates had similar and slightly alkaline optimal pH values, ranging from pH 7.5–8.5 (Fig. 4b, c). Overall, MBFV-derived prM peptides were better substrates for furin than TBFV-derived substrates, and the optimal pH for cleavage of these substrates by furin protease was neutral to slightly alkaline. These data suggest that the direct interaction between furin protease and furin cleavage site (or the adjacent region) is not pH-dependent, and does not cause differences in maturation between TBFV and MBFV (Fig. 4a–c).

Next, we examined the steric accessibility of the furin site due to various interactions within prM-E proteins. We used available sequences and AlphaFold modelling to investigate interactions within the prM-E hetero tetramer complexes of TBEV and USUV (Fig. 4d–g and Supplementary Fig. 5). Interestingly, the furin cleavage site in the linker between the transmembrane and globular part of the prM protein (Fig. 4d) was buried in the mature fully smooth conformation of the prM-E tetramer complex, indicating low accessibility to furin protease (Fig. 4e).

A detailed view of the prM cleavage site and E protein interface of TBEV showed that residue E83 (three residues upstream of the RTRR cleavage site) makes the three positive charges in the RTRR cleavage motif less dependent on the single negative charge of E243 in the E protein that interacts with the RTRR cleavage site (Fig. 4f). In contrast, K88 (one position upstream of the furin cleavage site RSRR) of USUV makes the three positive charges in RSRR in prM more dependent on the two negative charges of E241 and E243 in the E protein that interact with the RSRR site. Thus, the furin cleavage site shows a stronger interaction with the E protein, and prevents USUV maturation, at pH 7 (Fig. 4g). This is different from TBEV, where the E83 in prM can replace the single E243 in the E protein. In addition to the ionic interactions between prM and E proteins involving the furin cleavage site, we hypothesized that K81 in the prM and N256 in the E protein of TBEV are important for prM-E geometrical compatibility, based on structural analysis of the quaternary models.

These data suggest that E243 is important in TBEV maturation. Differences in interactions within the cleavage site between TBFV and MBFV indicate that uncleaved prM protein causes steric clashes that disrupt the prM-E quarternary complex, and prevent formation of the fully smooth conformation in the Golgi complex with acidic pH or neutral pH, after release from the host cell (Fig. 4h).

TBEV amino acid residue mutations elucidate the intermediate stage of flavivirus maturation

To confirm that the differences in amino acid interactions within the prM-E complex in USUV versus TBEV were related to virus immaturity and infectivity, we performed site-directed mutagenesis to introduce three amino acid substitutions into the TBEV genome (Supplementary Figs. 6, 7 and 8). The recombinant TBEV (m-rTBEV) carried one mutation in the E protein (N256A) and a double mutation in the prM protein: K81G (potentially relevant to the conformational compatibility of RTRR with E protein), and E83R (relevant to the RTRR pH dependency on E protein E243). Comparing the plaque morphology of m-rTBEV with m-TBEV revealed no differences in plaque size (Fig. 5a). Next, the replication kinetics in PS cells showed a peak at 48 hpi, and reached a titre of 2.7 × 10⁷ for m-rTBEV, and 5.9 × 10⁷ PFU ml^− 1 for m-TBEV (P < 0.01) (Fig. 5b). Interestingly, the growth kinetics in the tick cell line IRE/CTVM19 showed a more pronounced attenuation at 10 dpi: titre of 5.1 × 10⁴ PFU ml^− 1 for m-rTBEV, compared to 3.2 × 10⁵ PFU ml^− 1 for m-TBEV (P < 0.01) (Fig. 5c). Overall, m-rTBEV exhibited slightly reduced growth ability compared to m-TBEV in mammalian cell lines, but a greater attenuation when infecting tick cells.

Next, we infected LoVo cells to produce an immature variant of rTBEV (prM-rTBEV). At 48 hpi, prM-rTBEV reached a titre of 6 × 10³ PFU ml^− 1, compared to 3 × 10⁵ PFU ml^− 1 for prM-TBEV (Fig. 5d). The RNA levels (Fig. 5d) resulted in a genome equivalent:PFU ratio of ~ 27 for prM-rTBEV compared to ~ 2 for prM-TBEV (P < 0.0001) (Fig. 5e). These data indicated a shift in the specific infectivity of the recombinant immature variant towards that of MBFV. To assess the pH dependence of prM-rTBEV, the virus was pretreated with r-furin and then PS cells were infected. The virus titre of prM-TBEV barely changed, while the virus titre of prM-rTBEV was lower at neutral pH compared to acidic pH, suggesting that furin cleavage was more efficient with prM-rTBEV at acidic pH (P < 0.01) (Fig. 5f).

To obtain more detailed information, we incubated the virus with r-furin and then immediately performed a plaque assay. Immature prM-rTBEV showed a slightly reduced efficiency of furin cleavage at pH 7.5 compared to an acidic pH (P < 0.01), indicating a change in sensitivity similar to that of MBFV. Interestingly, no differences were observed between prM-TBEV samples incubated at acidic and neutral pH, but cleavage by endogenous furin during the plaque assay was more efficient at neutral pH compared to acidic pH (P < 0.01) (Fig. 5g). This trend in cleavage efficiency was opposite to our observations with prM-rTBEV. Overall, these results suggest that prM-rTBEV showed maturation properties generally similar to those of prM-USUV (MBFV). This supports our hypothesis that E243 is important for prM-TBEV maturation, and helps us understand the interactions in the prM-E protein complex during intermediate stages (Fig. 5h) of flavivirus maturation.

MBFV and TBFV have been thought to share fundamental molecular and structural properties, with correspondingly similar replication and maturation strategies within host cells²⁸. However, our present findings reveal crucial disparities between the maturation processes of these two virus groups. Previous studies of immature flaviviruses have predominantly focused on structural characteristics, particularly the architecture of the spiky immature particle^{26, 32, 33, 34}. In contrast, here we explored functional characteristics resulting from structural discrepancies, emphasizing an intermediate virus variant—an immature virion presenting a smooth surface with uncleaved prM protein. This model elucidates aspects of the intermediate phases of maturation.

Strikingly, our research revealed substantial differences in infectivity between prM-containing virions of TBFV versus MBFV. The infectivity of fully mature versus prM-containing TBFV differed by a maximum of 10-fold, while this discrepancy was significantly greater (1,000- to 10,000-fold) between mature versus prM-containing MBFV. Immature DENV (a typical MBFV) was previously described as infective in the presence of antibody-dependent enhancement (ADE), enabling otherwise non-infectious immature virus to enter the cell^{35, 36, 37}. However, we found that immature TBFV were infectious even without ADE.

Our investigations confirmed that furin cleavage was necessary in all cases, consistent with previously described mechanisms in TBEV and other flaviviruses³⁸. Moreover, we found that furin cleavage was not limited to virus production, but also occurred during virus entry into the host cell. Earlier assumptions suggested that final virus maturation might occur upon host cell entry, under specific conditions^{31, 39}. For example, the insect-specific flavivirus Binjari virus is exclusively produced as an immature variant within host cells and furin cleavage occurs upon subsequent host cell entry⁴⁰.

Previous studies of TBEV maturation and immature virus particle production have primarily utilized techniques that inhibit Golgi complex acidification, for example with ammonium chloride or bafilomycin A1^{19, 41, 42, 43}. This is done to impede conformational alterations that would render the prM cleavage site accessible to furin, thus preventing cleavage. These immature TBEV particles lack fusion activity due to the presence of uncleaved prM protein, which effectively obstructs fusion loop protrusion⁴⁴. Consequently, prM functions as a chaperone, preventing virions from engaging in fusion within the host cell’s acidic environment before their release²². In contrast, here we produced prM-TBEV particles by infecting furin-deficient LoVo cells, a well-established system for generating smooth immature flavivirus particles³⁰.

Even after furin-mediated cleavage, the "pr" portion of prM remains partially associated with the virion. The “pr” peptide only dissociates from the virion upon exposure to neutral pH in the extracellular milieu. This dissociation allows virions to unveil the E protein fusion loop upon encountering acidic pH, typically within the endosome of a newly infected cell^{20, 23, 24}. This process highlights the role of prM role in modulating the fusion activity of TBEV virions, ensuring that viral fusion occurs at the appropriate cellular location and time, ultimately influencing viral infectivity and propagation.

Our findings indicated that uncleaved prM does not impede the binding of smooth prM virions to the host cell surface. Moreover, furin on the cell surface enabled prM cleavage, facilitating further virus maturation after binding. We hypothesize that the neutral pH of the cell surface enables dissociation of the "pr" portion of prM-TBEV, allowing virion transition into an infectious state upon entry. This suggests that the cell surface-associated furin protease is critical for maturation, as immediate “pr” dissociation upon binding may contribute to rapid virion transition into an infectious state. The scenario differs for MBFV, as prM-USUV did not exhibit infectivity at neutral pH, even with r-furin. This observation highlights distinctive behaviours of MBFV and TBFV during maturation.

Numerous attempts have been made to mutate the furin cleavage site in TBEV^{45, 46, 47} and other flaviviruses^{43, 48, 49}. Mutants lacking the furin cleavage site or exhibiting altered cleavage specificity have encountered substantial evolutionary pressure, promoting restoration of the wild-type variant⁴⁵. Recent investigations of JEV and ZIKV with mutations in or near the furin cleavage site have demonstrated notable changes in replication and virulence^{48, 50}. The outcomes of the present study are consistent with existing literature, highlighting that residues within and near the cleavage site play critical roles in modulating specific interactions with furin protease⁵¹.

Furthermore, we demonstrated that MBFV-derived peptide substrates exhibited higher cleavage efficiency than TBFV-derived peptides, but a neutral pH was optimal for all tested substrates. Consequently, the differing pH sensitivity between TBFV and MBFV likely arises from steric interactions within the dimeric prM-E complex. This discrepancy underscores the importance of these complex interactions in dictating the pH sensitivities observed in the two flavivirus groups.

Previous research has extensively investigated the prM-E complex and its pivotal roles in fusion machinery activation, viral particle assembly, and pH-triggered structural changes in flaviviruses^{21, 24, 42, 48, 52, 53}. A single mutation in TBEV prM E88G (corresponding to E87 or E83 as subsequently reported³⁸) was found to have no impact on virus-like particle production⁴⁷, corroborating our findings. Similarly, in DENV, two histidine residues have been identified as a "toggle switch" controlling virus maturation. Specifically, the histidine residue at position 98 within the M region of prM is crucial for releasing the M segment from the E protein at low pH. Low pH induces increased protonation of H98 in prM, and H244 in the E protein, leading to electrostatic repulsion between the M part and the E protein. This causes enhanced binding of “pr” to E, facilitating conversion of the spiky trimer into a smooth dimer conformation²⁴.

Recent research on various mutations in the prM and E proteins of ZIKV demonstrated that E244 mutation in the E protein reduced viral titres and altered specific infectivity, underscoring the importance of this residue in viral maturation⁴⁸. However, the authors suggested that furin cleavage occurs after transition to the smooth conformation. In contrast, our findings suggested that furin cleavage in prM-TBEV occurred before attaining the fully smooth conformation, based on the inaccessibility of the furin cleavage site within the centre of the E dimer. Therefore, we suggest that furin cleavage occurs during an intermediate conformational state, before final fixation into a fully smooth structural rearrangement, and residues remaining after cleavage must dissociate from the E protein to prevent steric clashes and allow complete maturation. Recent findings based on cryoEM structural analysis of spiky immature TBEV support this, proposing that prM cleavage must precede the formation of the herringbone smooth structure, as the furin cleavage site would otherwise be inaccessible²⁷.

Furthermore, our structural modelling of prM-TBEV implied that E83 of prM diminished the dependency of positive charges within the furin cleavage site on the negative charge of E243 in the E protein, elucidating the crucial role of E243 in prM-TBEV maturation. The limitation of our approach lies in its reliance on modelling, as structural data on the smooth immature TBEV could offer deeper insights. However, the structural study of immature spiky TBEV highlighted the significance of residues E243 and R88 from the E and prM proteins, respectively. These residues, along with the nearby hydrophobic zipper region, stabilize the furin cleavage site within the prM-E complex. This provides further support for our modelling and mutagenesis results²⁷. In contrast, prM-USUV exhibited an even stronger prM-E interaction due to K88 of prM, heightening the dependence of positive charges at the furin cleavage site on negative charges of E-protein residues E241 and E243. This impedes prM-USUV maturation at neutral pH. Using structural modelling and experimental validation, we demonstrated that recombinant prM-rTBEV (with mutations E83R and K81G in prM, and N256A in E) displayed less efficient furin cleavage, yielding a higher proportion of uncleaved virus particles. Additionally, the pH sensitivity shifted closer to that of prM-USUV, or generally towards MBFV. However, one limitation of our approach is that it cannot validate whether the introduced mutations directly affect the pH-dependent binding between prM and the E protein. In addition, we cannot rule out the possibility that the mutagenesis experiments on the E protein conducted in this study could lead to the destabilization of the E dimer, making it more similar to those of MBFV. The ectodomain of the flavivirus E protein demonstrates that the dimer in TBFV is much more stable than in MBFV ¹⁸. This increased stability could explain the irreversibility of the spiky-to-smooth transition: once the E dimers form, they do not dissociate, with the pr portion tethered via the uncleaved furin site, which can be cleaved by furin during entry. In contrast, the more labile E dimer of MBFV dissociates reversibly, allowing the spiky form to reform when exposed to neutral pH if prM remains uncleaved.

During infection at acidic pH, immature prM-USUV assumes an intermediate smooth conformation, making the cleavage site accessible. We hypothesize that upon host cell binding, prM-USUV undergoes cleavage on the cell surface, enabling virion entry into host cells. Subsequently, within early endosomes at pH of ~ 6.5, we postulate that the "pr" segments dissociate from the virions. This triggers fusion machinery activation, facilitating membrane fusion within the late endosomes (pH 5.5), ultimately allowing cell infection.

Numerous investigations of TBFV have relied on extrapolation from studies of MBFV, which have been more extensively researched. However, our study highlights profound discrepancies in intermediate maturation stages between these two groups, emphasizing the critical need for independent and in-depth examinations of these viruses. Moreover, it underscores the likelihood that distinct evolutionary pressures act on these viruses within hosts and vectors, potentially leading to differing maturation strategies and biological behaviours⁵⁴. A comprehensive analysis of TBFV is imperative to elucidate their unique characteristics and evolutionary adaptations, offering valuable insights into their infectivity, transmission, and pathogenicity.

In conclusion, our results significantly diverge from the traditional understanding of flavivirus maturation, highlighting marked differences between MBFV and TBFV. Our focus on functional aspects, particularly emphasizing an intermediate virus variant with an uncleaved prM protein, challenges the belief that immature flaviviruses exhibit uniformly low infectivity. We also propose a new perspective on furin cleavage during virus entry, revealing distinct infection patterns between MBFV and TBFV. These findings emphasize the need for tailored investigations of each virus group, avoiding generalized assumptions derived from studying MBFV. Understanding the contributions of diverse virion populations is pivotal for unravelling flavivirus transmission dynamics and pathogenicity, potentially guiding targeted antiviral strategies against flavivirus infections, and elucidating evolutionary pressures shaping these virus groups.

Virus strains

TBFV were represented by TBEV strain Hypr (of the European subtype), LIV (strain LI/31) and LGTV (strain TP-21), which were all provided by the Collection of Arboviruses, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice. MBFV used in this study included ZIKV (Brazilian strain Paraiba_01), kindly provided by Prof. Paolo M. de A. Zanotto, University of São Paulo, Brazil; USUV (Eu1 lineage strain 200/TM/10), provided by the Collection of Arboviruses, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice⁵⁵; and WNV (strain 13–104, representative of genomic lineage 2, isolated in the Czech Republic), kindly provided by Prof. Zdenek Hubalek and Dr. Ivo Rudolf, Institute of Vertebrate Biology of the Czech Academy of Sciences⁵⁶.

Mammalian and tick cell lines

Porcine kidney stable PS cells (National Reference Laboratory for Cell Cultures, National Institute of Public Health, Prague) were grown in Leibovitz’ L-15 medium (L-15). Golden hamster BHK-21 (ATTC CCL-10) and African green monkey Vero cells (ATCC CCL-81) were grown in Dulbecco’s modified Eagle’s medium (DMEM). Furin-deficient human LoVo cells (human colon carcinoma, ATCC CCL-229) were grown in Ham’s F12 medium. Cells were cultured in media supplemented with foetal bovine serum (3% in L15, 10% in DMEM, or 20% in Ham's F12), 100 U ml^− 1 penicillin, 100 µg ml^− 1 streptomycin, and 2 mM glutamine (Sigma-Aldrich, Prague, Czech Republic), at 37°C in 5% CO₂ (except for PS cells, which were cultured without extra CO₂). The tick cell line IRE/CTVM19⁵⁷ (derived from Ixodes ricinus, Tick Cell Biobank, University of Liverpool, UK) was maintained at 28°C without extra CO₂, in L15 medium supplemented with 10% foetal bovine serum and 5% tryptose phosphate broth, as previously described⁵⁸.

Production of prM-containing immature particles

Immature prM-containing virus particles were produced using LoVo cells, which are deficient in furin protease⁵⁹, and thus provide an easy-to-use platform for immature virus production without affecting the pH within the maturation pathway. For propagation, cells were seeded in 96-well plates (approximately 3 × 10⁴ cells per well), and incubated at 37°C under 5% CO₂ for 24 hours, to form a confluent monolayer. Then these cells were infected with viruses at a multiplicity of infection (MOI) of 2. After 12 h, the virus suspension was removed, the cells were washed three times with phosphate-buffered saline (PBS), and fresh medium was added. At 48 h post-infection (hpi), the virus was harvested, and viral titre was determined by plaque assays, or viral RNA copies were quantified by quantitative PCR with reverse transcription (RT-qPCR), as described below.

Plaque assay

Plaque assays were performed as previously described⁶⁰, with slight modifications. Briefly, viruses were diluted 10-fold in 24-well tissue culture plates, and 1–1.5 × 10⁵ PS or Vero cells were added to each well. After 4 h of incubation, the wells were covered with 1.5% (w/v) carboxymethylcellulose (CMC) in L-15 (PS cells for TBEV, LGTV, LIV, and USUV) or DMEM (Vero cells for ZIKV and WNV) medium. After five days of incubation at 37°C, infected plates were washed with PBS, and cell monolayers were stained with naphthol blue-black. Viral titre was expressed as plaque-forming units (PFU) per ml.

RNA isolation, RT-qPCR, RT-PCR, and sequencing

Viral RNA was isolated from culture supernatants using the QIAmpViral RNA mini kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. RT-qPCR was performed on a LightCycler 480 II in a 96-well plate block (Roche, Basel Switzerland), using the Advanced Kits for Tick-borne Encephalitis, Zika Virus, or West Nile Virus (Genesig, Germantown, MD, USA) or the Usutu Virus Kit (#FR439; Genekam Biotechnology AG, Duisburg, Germany). Lyophilized OneStep qRT-PCR master mix (Oasig) was used, following the manufacturer’s instructions. For TBEV, ZIKV, and WNV, the copy numbers per µl were calculated from calibration curves based on standards provided by the manufacturer. For USUV, the calibration curve was developed using serial dilutions of RNA isolated from a virus stock with a known titre in PFU ml^− 1; therefore, the qRT-PCR results for USUV were presented in relative PFU per ml (rPFU ml^− 1). For sequencing, the isolated RNA was reverse-transcribed using a QIAGEN OneStep Ahead RT-PCR Kit (Qiagen, Germantown, MD, USA). Amplified products were separated in 1% agarose gel, and purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA). Sanger sequencing was performed by the commercial provider Eurofins Genomics.

Plaque-reduction assay

PS cells were seeded in 6-well tissue culture plates (0.8–1 × 10⁶ cells per well), and incubated for 24 h. Confluent cells were pretreated for 2 h with the furin inhibitor Decanoyl-RVKR-CMK (Sigma Aldrich), and then infection was performed using 50 PFU per well, at 37°C for 1 h. Next, the unbound virus was removed, the cells were washed three times with PBS, and fresh medium with a CMC overlay was added. After incubation at 37°C for five days, infected plates were washed with PBS, and cell monolayers were stained with naphthol blue-black as described for the plaque assay.

Virus in vitro furin cleavage assay

The prM-TBEV sample was concentrated using Amicon ultra-centrifugal 100-kDa filters, and medium-buffer exchange was performed by centrifugation (4,500 rpm, 4°C, 7 min; TX-400 rotor, 75003629; Thermofisher Scientific). Next, the medium was changed to Tris-maleate buffer²⁰ (TMB, 50 mM) containing CaCl₂ (10 mM), with the pH adjusted to 5.5 or 7.5. For furin in vitro cleavage, 1 µl of recombinant human furin (r-furin, #SRP6274; Sigma Aldrich) was added to the 100-µl reaction, followed by incubation at 37°C for 2 h. Next, LoVo cells in TMB were incubated with the virus (MOI 0.1) for 4 h. Then the unbound virus was removed, the cells were washed three times with PBS, and fresh medium was added. Finally, at 48 hpi, the viral progeny was harvested for further analysis. Infection with prM-USUV was performed using PS cells, with the same in vitro cleavage procedure as described above.

Fluorescent infectious assay

Recombinant mCherry-expressing TBEV was used to visualize viral infection⁶¹. PS cells were cultured in 96-well plates, and infected as described above. At 48 hpi, nuclei were stained with Hoechst 33342 (Invitrogen), following the manufacturer’s recommended protocol. Immunofluorescence of prM-TBEV and prM-USUV infection was visualised after fixation in ice-cold methanol-acetone mixture (1:1) with primary anti-flavivirus protein E antibodies (1:250; Sigma Aldrich) and then with secondary antibodies AlexaFluor 488 (1:1000; Thermofisher Scientific) and DAPI (1:2000; Sigma Aldrich) to stain cell nuclei. Images were acquired using an Olympus IX81 epifluorescence microscope, equipped with Olympus 10× UPLFLN lenses, and a Hamamatsu OrcaR2 camera controlled by Olympus Xcellence software. Raw images were processed using ImageJ/Fiji software.

Western blot analysis

To investigate prM cleavage by furin, we performed western blot analysis. Virus samples used for western blot analysis were produced in medium mixture DMEM:HAM F12 (1:1) supplemented with a reduced FBS concentration (2%), and concentrated using Amicon ultra-centrifugal 100-kDa filters. Laemmli sample buffer was mixed 1:3 with the virus sample, and incubated for 5 min at 95°C. After cooling on ice, samples were ready for downstream analysis. Proteins were separated by standard SDS-PAGE, and then transferred to an Immobilon®-P PVDF Membrane (Millipore). Blots were blocked overnight at 4°C in blocking buffer comprising PBS, 0.05% Tween 20, and 2% Amersham ECL Prime Blocking Reagent (Cytiva). Next, the blots were stained with primary antibodies against TBEV E protein (mouse monoclonal antibody 1493⁶² diluted 1:2000) or M protein (in-house rabbit polyclonal serum⁶³ diluted 1:500), overnight at 4°C, followed by the secondary antibodies anti-mouse IgG-HRP (Invitrogen AB_228307) or anti-rabbit IgG-HRP (Invitrogen AB_228341) for 1 h at room temperature. Finally, blots were visualized on an Amersham™ Imager 680 (GE Healthcare).

Mouse infection

We evaluated the infectivity of prM-containing viruses in 6-week-old female BALB/c mice (ENVIGO RMS). Six groups of mice were inoculated s.c. with TBEV or prM-TBEV strain Hypr as follows: group 1 (n = 5), TBEV 10³ genomic equivalents (gen. eq.) per mouse; group 2 (n = 10), TBEV 10² gen. eq. per mouse; and group 3 (n = 5), TBEV 10¹ gen. eq. per mouse, respectively. Groups 4–6 were infected with the equivalent doses of the prM-TBEV variant. For comparison with MBFV, WNV strain 13–104 was used and the experimental set-up and mouse groups were the same as in the TBEV experiment. Over a 28-day experimental period, the survival rates and symptoms were monitored daily. Symptoms were evaluated using the following clinical scores 1 = healthy, 2 = piloerection, 3 = hunched back, 4 = paralysis, and 5 = death. Changes in body weight upon infection were monitored for 14 days. All mice exhibiting a clinical score of 4 were humanely terminated (by cervical dislocation) immediately upon detection of symptoms.

Blood was taken 3 dpi from the tail vein to evaluate the virus titre in the serum. After dissection of the brain on day 8, tissue samples were homogenized in DMEM medium (+ 10% FBS) to obtain a 20% suspension. After centrifugation (5000 rpm, 4°C, 10 min; TX-400 rotor, 75003629; Thermofisher Scientific), the supernatant from suspension was used for the plaque assay.

Protease fluorescent assay

Four internally quenched fluorescent substrates—Dabcyl-EGSRSRRSVL-Edans, Dabcyl-EGSRTRRSVL-Edans, Dabcyl-HSRRSRRSLT-Edans, and Dabcyl-HSKRSRRSIA-Edans—were synthesized without C-terminal Edans, using a standard solid phase peptide synthesis protocol⁶⁴, with 2-chlorotrityl chloride resin support. The N-Dabcylated side chain protected peptides were released from the resin using a mixture of acetic acid/2,2,2-trifluoroethanol/dichloromethane (1:1:3) for 2 h, and then were evaporated and dried. Next, the Edans was introduced via Edans acid sodium salt (1.5 eq. to peptide) and PyBOP (2.0 eq. to peptide) in the presence of DIPEA (3 eq. to peptide) in DMF as a solvent, overnight. After solvent evaporation, side chains were deprotected using a mixture of TFA/triisopropylsilane/water (95:2.5:2.5) for 1 h, and then liquids were evaporated. The residues were purified by preparative RP HPLC, and characterised by LC/MS-ESI: Dabcyl-EGSRSRRSVL-Edans [M + H]⁺ 1645.8, Dabcyl-EGSRTRRSVL-Edans [M + H]⁺ 1659.8, Dabcyl-HSRRSRRSLT-Edans [M + H]⁺ 1754.8, and Dabcyl-HSKRSRRSIA-Edans [M + H]⁺ 1696.9.

Lyophilized human recombinant furin protease produced in Hi-5 insect cells (PeproTech) was diluted to 1 mg ml^− 1 using water, according to the manufacturer’s instructions. The assay buffer of an appropriate pH contained 25 mM acetic acid, 25 mM 2-[morpholino]ethanesulfonic acid (MES), 25 mM glycine, and 1 mM CaCl₂⁶⁵. Fluorescence measurements were performed in 96-well Greiner chimney black plates. All assays were started by adding furin to a final concentration of 10 ng mL^− 1 in the 100-µl reaction volume. The time-dependent increase of fluorescence upon enzyme addition was monitored for 30 min, with a kinetic cycle of 1 min, using a Tecan Spark plate reader with an excitation wavelength of 336 nm and emission wavelength of 490 nm. All measurements were made in triplicate.

Quaternary structure analysis

For both TBEV and USUV, models of the prM-E complexes (containing the complete protein sequences of the prM and E proteins) were made using AlphaFold2⁶⁶. These were used to generate the quaternary structure model of a dimer of heterodimers, based on the PDB code templates 7Z51, 5O6A, 7LCH, and 7QRE^{18, 38, 63, 67}. Structural analysis of the models was performed in PyMOL 2.5.4. The quaternary models were used to predict mutations that would potentially affect the infectivity of viruses, which were modelled with AlphaFold2 to check the effects on the quaternary structure of TBEV and USUV, particularly the proximity of the furin cleavage site.

Site-directed mutagenesis and transfection

The reverse genetics system used was based on the generation of three infectious subgenomic overlapping DNA fragments encompassing the whole TBEV genome (strain Hypr), as previously described⁶⁸. Fragments I and III were flanked by the human cytomegalovirus promoter (pCMV) and hepatitis delta ribozyme, followed by the simian virus 40 polyadenylation signal sequence (HDR/SV40pA). All three fragments were synthesized and individually cloned into pUC57 or pC11 vectors (GenScript, Piscataway, NJ, USA).

To produce genomic fragments with mutations in prM and E proteins, fragment I was used as a template for amplification with modified primers (Supplementary Fig. 8, Supplementary Table 1). This enabled production of two sub-fragments, Ia and Ib, each with overlapping homology arms. PCR and fragment purification were performed as previously described⁶¹.

For transfection, BHK-21 cells were seeded in a 24-well plate at a density of 3 × 10⁵ cells (400 µl) per well, and incubated overnight. Cell transfection was performed using an equimolar mixture of the four DNA fragments. A DNA-lipid complex was prepared using X-tremeGENE Transfection Reagent and 200 µl Opti-MEM (Thermofisher Scientific), followed by a 15-min incubation at room temperature. The entire mixture was added drop-wise to the well with the cell monolayer, and then incubated for 4 days. Then the supernatant was collected, and viral titre was determined by plaque assay. Finally, mutagenesis was confirmed by sequencing.

Statistics, reproducibility, and graphics

GraphPad software Prism v.8 was used for statistical analysis. In all figures, data were plotted as means with error bars denoting the SD. All statistical tests were performed using the Mann-Whitney nonparametric test. In animal experiments, the survival rates were statistically evaluated using the log-rank Mantel-Cox test. The significance threshold was set at P < 0.05. Figures 1a, 2h, 5h, and Supplementary Fig. 8 were created with BioRender.com.

Ethics statement

The animal experiments complied with all relevant European Union guidelines for work with animals, and were conducted in accordance with Czech national law guidelines on the use of experimental animals and protection of animals against cruelty (Animal Welfare Act No. 246/1992 Coll.). The protocol was approved by the Committee on the Ethics of Animal Experimentation of the Institute of Parasitology, and the Departmental Expert Committee for the Approval of Projects of Experiments on Animals of the Czech Academy of Sciences (permit no. 111/2020).

Author contributions

J.H. and D.R. conceived and designed the study and wrote the manuscript. J.H., J.S., M.M., P.B., P.N., M.H., T.M., E.R., L.E., A.F., M.B., A.Ö, A.C. performed the analyses. L.B.-S. contributed essential materials. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

Inclusion & ethics

All collaborators of this study have fulfilled the criteria for authorship required by Nature Portfolio journals have been included as authors, as their participation was essential for the design and implementation of the study. Roles and responsibilities were agreed among collaborators ahead of the research.

Acknowledgements

This study was supported by the Czech Science Foundation (grant No. 23-07160S, awarded to D.R.), the EEA and Norway Grants Fund for Regional Cooperation (project number 2018-1-0659, awarded to D.R.); and the National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22N-PO5103), funded by the European Union—Next Generation EU. This study was also supported by Charles University Grant Agency (project no. 50121). L.B.S. was supported by the Wellcome Trust (grant no. 223743_Z_21_Z). A.K.Ö was supported by the Swedish Research Council (grant no. 2020-06224). We thank Jan Konvalinka and Milan Kožíšek for fruitful discussions.

Data availability

All data supporting the findings of this study are available within the article and its supplementary files. Any additional requests for information can be directed to, and will be fulfilled by, the corresponding author.

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Divergent pathogenic dynamics of immature tick-borne and mosquito-borne flaviviruses: a paradigm shift in prM-containing particle infectivity

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Abstract

Figures

Introduction

Results

Production and characterization of prM-TBEV

Furin-mediated cleavage during virus entry

Immature prM-flavivirus infectivity in TBFV and MBFV

pH-dependent furin cleavage in TBEV and USUV

Pathogenicity of prM-TBEV and prM-WNV in a mouse model

Structural prM-E interface and its effect on furin cleavage site accessibility

TBEV amino acid residue mutations elucidate the intermediate stage of flavivirus maturation

Discussion

Methods

Virus strains

Mammalian and tick cell lines

Production of prM-containing immature particles

Plaque assay

RNA isolation, RT-qPCR, RT-PCR, and sequencing

Plaque-reduction assay

Virus in vitro furin cleavage assay

Fluorescent infectious assay

Western blot analysis

Mouse infection

Protease fluorescent assay

Quaternary structure analysis

Site-directed mutagenesis and transfection

Statistics, reproducibility, and graphics

Declarations

References

Additional Declarations

Supplementary Files

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