Disruption of Autographa Californica Multiple Nucleopolyhedrovirus ac111 Results in Reduced per os Infectivity in a Host-Dependent Manner

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac111 gene is highly conserved in lepidopteran-specific baculoviruses, and its function in the AcMNPV life cycle is still unknown. To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner.


Introduction
Baculoviruses are a family of rod-shaped, enveloped, double-stranded DNA viruses which are specifically pathogenic to invertebrate species, mainly belonging to the orders Lepidoptera, Hymenoptera, and Diptera [1]. Baculoviridae consists of Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus [2]. Based on phylogenetic analysis of the polyhedrin (polh) gene, Alphabaculovirus can be further subdivided into Group I and Group II nucleopolyhedroviruses (NPVs) [3]. Autographa californica multiple NPV (AcMNPV) is the archetype of the Baculoviridae family and the first baculovirus to be completely genome-sequenced [4]. The AcMNPV genome is approximately 134 kbp and encodes 154 potential open reading frames (ORFs) [4]. To date, more than 80 baculovirus genomes have been completely sequenced, and there are 38 genes conserved in all these genomes. These 38 common genes are considered to be the baculovirus core genes [1,[5][6][7].
During the baculovirus infection cycle, two morphologically distinct but genetically identical progeny virion phenotypes, budded virions (BVs), and occlusion-derived virions (ODVs), are produced. Although BVs and ODVs are similar in their nucleocapsid structure, they differ in their origin and the composition of their envelopes, which reflects their distinct roles in the baculovirus life cycle [8,9]. In the early infection phase, newly assembled nucleocapsids egress from the nucleus, migrate across

Construction of an ac111-Knockout Virus
Employing the bacmid bMON14272, an ac111-knockout AcMNPV bacmid (bAc111KO) was generated through ET homologous recombination in Escherichia coli, as previously described [44]. Briefly, a 400-bp fragment that was homologous to the 5 region of the ac111 ORF (AcMNPV nt 101,822 to 102,221) was PCR-amplified from bMON14272 using the primers ac111PF1/ac111PR1 (the PCR primers used in this study are listed in Table 1). A 340-bp fragment homologous to the 3 region of the ac111 ORF (AcMNPV nt 101,402 to 101,741) was PCR-amplified from bMON14272 using the primers ac111PF2/ac111PR2. The 5 -and 3 -PCR products were digested with PstI/HindIII and SacI/BamHI, respectively, and ligated into pUC18-Cm [45], which contains a chloramphenicol resistant (Cm) gene cassette to generate pUC18-111US-Cm-111DS. The plasmid was then digested with SacI and HindIII to obtain a linear 1778-bp fragment in which the Cm gene cassette was flanked by ac111 5 -and 3 -regions. Homologous recombination between the deletion region of ac111 (AcMNPV nt 101,742 to 101,821) and the Cm cassette in the linear fragment was performed to obtain an ac111-knockout bacmid, bAc111KO. The replacement in bAc111KO was confirmed by PCR and sequencing. Table 1. Primer sequences used in this study.

Applications Primer Sequence
Amplification of ac111 5 flanking sequences ac111PF1 5 -GAGCTCGTTTCCATAATAATGAGGATACGCT-3 To facilitate the detection of viral infection, the pFB1-PH-GFP donor plasmid, containing the AcMNPV polh gene and the enhanced green fluorescence protein (egfp; referred to as gfp in the present study) gene, was transformed into electrocompetent DH10B cells harboring the bAc111KO bacmid and the pMON7124 helper plasmid as previously described [45], to generate the ac111-knockout virus, vAc111KO. To generate the ac111-repaired virus, vAc111:HA, the donor plasmid pFB1-Ac111:HA-PH-GFP was constructed using the following steps. The 531-bp DNA fragment containing the promoter and the ORF of ac111 tagged with an HA epitope prior to the stop codon was PCR amplified from bMON14272 using the primers ac111PF3/ac111PR3, and it was cloned into the SacI/BamHI-digested pUC18-SV40 plasmid [46] to construct the pUC18-Ac111:HA plasmid. The Ac111:HA fragment was digested from pUC18-Ac111:HA with SacI/XbaI, and subcloned into SacI/XbaI-digested pFB1-PH-GFP to generate the donor plasmid pFB1-Ac111:HA-PH-GFP. The pFB1-Ac111:HA-PH-GFP was then transformed into electrocompetent DH10B cells harboring the bAc111KO bacmid and the helper plasmid pMON7124 to generate the ac111-repaired virus, vAc111:HA. Similarly, vAcWT, a wild type virus, was also constructed by inserting polh and gfp genes into bMON14272.

Real-Time PCR (qPCR) Analysis
qPCR analysis was performed to assess the effect of the ac111 deletion on viral DNA synthesis as described previously [7,46].

Transmission Electron Microscopy (TEM)
1.0 × 10 6 Sf9 cells were transfected with 1.0 µg bacmid DNA of vAc111KO, vAc111:HA, or vAcWT. At 72 h p.t., the cells were dislodged with a cell scraper, pelleted at 1000 × g for 10 min, and prepared for TEM, as previously described [7]. The samples were observed with a JEOL JEM-1400 transmission electron microscope (Tokyo, Japan) at an accelerating voltage of 120 kV.

Bioassays in T. ni and S. exigua Larvae
In order to determine whether the ac111 deletion had any effect on the infectivity of AcMNPV in insect larvae, bioassays were performed using T. ni or S. exigua larvae, as described previously [28]. Briefly, OBs and BVs were obtained from vAc111KO-, vAc111:HA-, or vAcWT-infected Sf9 cells, as previously described [48]. Different doses of BVs were hemocoelically injected into fourth-instar T. ni or S. exigua larvae to examine the infectivity of BVs in vivo. H 2 O was used as a blank control. For the oral infectivity bioassays, the OBs of vAc111KO, vAc111:HA, or vAcWT purified from infected Sf9 cells, were resuspended in double-distilled water and administered to newly molted T. ni or S. exigua larvae via oral inoculation. To determine the median lethal dose (LD 50 ), 1 µL water (control) or 1 µL OB suspensions of each virus was administered to a small piece of diet of newly molted third-instar T. ni or S. exigua larvae, respectively. For each virus in T. ni, the doses were 9, 30, 90, 3 × 10 2 , and 9 × 10 2 OBs per larva. For each virus in S. exigua, the doses were 9 × 10 2 , 3 × 10 3 , 9 × 10 3 , 3 × 10 4 , and 9 × 10 4 OBs per larva. Larvae were reared individually in 24-well plates until all of the diet was consumed, then fresh diet was added. Only the larvae that completely ingested the diet with OBs within 12 h were further reared and monitored daily, until all larvae had either pupated or died. The median lethal time (LT 50 ) was determined using the droplet feeding method [49]. The newly molted third-instar T. ni or fourth-instar S. exigua larvae were starved for 6 h, and then they were fed with 1 µL OB suspensions of each virus with concentrations of 1 × 10 7 or 1 × 10 8 OBs/mL, respectively. For each virus, the final mortalities were about 99% or 86% in T. ni or S. exigua, respectively. The larvae that have ingested virus were reared individually and the mortality was recorded every 12 h until all larvae had either pupated or died. The experiments with each dose in both oral and injection treatments were performed three times (30 larvae per dose). The LD 50 value and the LT 50 value were determined by probit analysis and the Kaplan-Meier estimator, respectively.
Viruses 2018, 10, x 5 of 16 in all Ac111 homologues ( Figure 1). Currently, no information about this motif in the function of ac111 is available and further studies need to be undertaken to determine its importance. Figure 1. Multiple-sequence alignment of 10 selected Ac111 homologues. The alignment was performed and edited using Clustal X and GeneDoc, respectively. The Ac111 homologue sequences used are as follows: NP_054141.

Construction of an ac111-Knockout AcMNPV
Based on AcMNPV transcriptomics [32], the 5′ transcription start site of ac110 is located 38 nt downstream of the 3′ end of ac111, and the 5′ transcription start site of ac112 is located 59 nt upstream of the initiation codon ATG of ac111. To investigate the role of Ac111 in the baculovirus life cycle, the ac111-knockout bacmid, bAc111KO, was constructed via the λ Red recombination system, as described previously [7]. In bAc111KO bacmid, 6 nt of the 5′-end and 118 nt of the 3′-end of ac111 ORF were retained to avoid affecting the transcription of ac110 and ac112; an 80-bp region of ac111 was deleted and replaced with a 1038-bp Cm cassette ( Figure 2A).
The absence of ac111 and the replacement with the Cm gene in AcMNPV bacmid were confirmed by PCR analysis. As shown in Figure2B, the primers ac111PF4/ac111PR2 produced no PCR product in bAc111KO, while a 420-bp fragment was amplified in AcMNPV bacmid bMON14272. Similarly, the primers ac111PF1/ac111PR4 produced no PCR product in bAc111KO but produced a 480-bp fragment in AcMNPV bacmid bMON14272. The primers CmF/CmR produced no PCR product in AcMNPV bacmid bMON14272, but a 1038-bp fragment was amplified in bAc111KO. Finally, the primers ac111PF1/ac111PR2 produced a 1778-bp fragment in bAc111KO but an 820-bp fragment in AcMNPV bacmid bMON14272. These results demonstrated that the target deletion region of ac111 was successfully replaced by the Cm gene.
In order to facilitate the observation of the viral infection progress, the polh gene of AcMNPV and the gfp gene were inserted into the polh locus of bAc111KO via Tn7-mediated transposition to construct the ac111-knockout AcMNPV, vAc111KO, as described previously [45]. To confirm the phenotype observed in vAc111KO resulted from the deletion of ac111, a rescue virus, vAc111:HA, was constructed by inserting the ac111 gene under the control of its native promoter with an HA tag prior to the ac111 stop codon, along with the polh and gfp genes, into the polh locus of bAc111KO ( Figure 2A). Similarly, a wild-type AcMNPV control, vAcWT, was generated by inserting the polh and gfp genes into bMON14272 (Figure 2A).

Ac111 is Not Required forBV Production
To study the effect of ac111 deletion on viral replication, Sf9 cells were transfected with vAc111KO, vAc111:HA, or vAcWT bacmid DNA, and virus infections were monitored using a fluorescence microscope. As shown in Figure 3A, similar amounts of GFP-positive cells were observed in the three groups at 24 h p.t., indicating comparable transfection efficiencies. By 72 h p.t., almost all vAc111KO-, vAc111:HA-, and vAcWT-transfected cells showed GFP fluorescence,

Construction of an ac111-Knockout AcMNPV
Based on AcMNPV transcriptomics [32], the 5 transcription start site of ac110 is located 38 nt downstream of the 3 end of ac111, and the 5 transcription start site of ac112 is located 59 nt upstream of the initiation codon ATG of ac111. To investigate the role of Ac111 in the baculovirus life cycle, the ac111-knockout bacmid, bAc111KO, was constructed via the λ Red recombination system, as described previously [7]. In bAc111KO bacmid, 6 nt of the 5 -end and 118 nt of the 3 -end of ac111 ORF were retained to avoid affecting the transcription of ac110 and ac112; an 80-bp region of ac111 was deleted and replaced with a 1038-bp Cm cassette (Figure 2A).
The absence of ac111 and the replacement with the Cm gene in AcMNPV bacmid were confirmed by PCR analysis. As shown in Figure 2B, the primers ac111PF4/ac111PR2 produced no PCR product in bAc111KO, while a 420-bp fragment was amplified in AcMNPV bacmid bMON14272. Similarly, the primers ac111PF1/ac111PR4 produced no PCR product in bAc111KO but produced a 480-bp fragment in AcMNPV bacmid bMON14272. The primers CmF/CmR produced no PCR product in AcMNPV bacmid bMON14272, but a 1038-bp fragment was amplified in bAc111KO. Finally, the primers ac111PF1/ac111PR2 produced a 1778-bp fragment in bAc111KO but an 820-bp fragment in AcMNPV bacmid bMON14272. These results demonstrated that the target deletion region of ac111 was successfully replaced by the Cm gene.
In order to facilitate the observation of the viral infection progress, the polh gene of AcMNPV and the gfp gene were inserted into the polh locus of bAc111KO via Tn7-mediated transposition to construct the ac111-knockout AcMNPV, vAc111KO, as described previously [45]. To confirm the phenotype observed in vAc111KO resulted from the deletion of ac111, a rescue virus, vAc111:HA, was constructed by inserting the ac111 gene under the control of its native promoter with an HA tag prior to the ac111 stop codon, along with the polh and gfp genes, into the polh locus of bAc111KO (Figure 2A). Similarly, a wild-type AcMNPV control, vAcWT, was generated by inserting the polh and gfp genes into bMON14272 (Figure 2A).

Ac111 is Not Required forBV Production
To study the effect of ac111 deletion on viral replication, Sf9 cells were transfected with vAc111KO, vAc111:HA, or vAcWT bacmid DNA, and virus infections were monitored using a fluorescence microscope. As shown in Figure 3A, similar amounts of GFP-positive cells were observed in the three groups at 24 h p.t., indicating comparable transfection efficiencies. By 72 h p.t., almost all vAc111KO-, vAc111:HA-, and vAcWT-transfected cells showed GFP fluorescence, indicating the infection had spread from the cells initially transfected. Light microscopy showed that OBs were produced in the three groups, and there were no obvious differences in the OB production ( Figure 3A), suggesting similar infection progress among all three viruses.
To further evaluate the effect of the ac111 deletion on virus propagation, viral growth curve analysis was performed. Sf9 cells transfected with vAc111KO, vAc111:HA, or vAcWT or infected with BVs showed a normal and steady increase in virus production and similar virus growth kinetics ( Figure 3B). In Se301 cells infected with vAc111KO, vAc111:HA, or vAcWT, the three virus growth curves were similar [50]. In addition, no obvious differences were observed in plaque sizes in Sf9 cells transfected with vAc111KO, vAc111:HA, or vAcWT [50].
Taken together, these results demonstrated that the deletion of ac111 had no apparent effect on viral replication in Sf9 cells or Se301 cells. indicating the infection had spread from the cells initially transfected. Light microscopy showed that OBs were produced in the three groups, and there were no obvious differences in the OB production ( Figure 3A), suggesting similar infection progress among all three viruses.
To further evaluate the effect of the ac111 deletion on virus propagation, viral growth curve analysis was performed. Sf9 cells transfected with vAc111KO, vAc111:HA, or vAcWT or infected with BVs showed a normal and steady increase in virus production and similar virus growth kinetics ( Figure 3B). In Se301 cells infected with vAc111KO, vAc111:HA, or vAcWT, the three virus growth curves were similar [50]. In addition, no obvious differences were observed in plaque sizes in Sf9 cells transfected with vAc111KO, vAc111:HA, or vAcWT [50].
Taken together, these results demonstrated that the deletion of ac111 had no apparent effect on viral replication in Sf9 cells or Se301 cells. The ac111 ORF has 204 nt. The first nucleotide of the initiation codon ATG of ac111 is indicated as +1. The 5′ transcription start site of ac110 is located 242 nt downstream of the 5′ end of ac111, and the 5′ transcription start site of ac112 is located 59 nt upstream of the initiation codon ATG of ac111. The ac111 deletion bacmid, bAc111KO, was generated by replacing an 80-bp fragment between the 6 and 87 nt of ac111 in the bMON14272 genome with a 1038-bp Cm cassette via ET homologous recombination. The polh and gfp genes were inserted into the polh locus of bAc111KO by Tn7-mediated transposition to construct the ac111 deletion virus, vAc111KO. The rescue virus, vAc111:HA, was constructed by inserting the ac111 gene under the control of its native promoter with an HA tag (indicated as a grey triangle) prior to the ac111 stop codon, together with the polh and gfp genes, into the polh locus of bAc111KO. The polh and gfp genes were inserted into bMON14272 to generate the wild-type control virus, vAcWT. The approximate locations of primers (arrows) used in the confirmation of the disruption of ac111 and the correct insertion of the Cm gene cassette are shown in the diagram; (B) Confirmation of bacmids by PCR. Bacmid DNA templates are shown above each

Ac111 is Not Required for Viral DNA Replication
To assess whether ac111 deletion leads to a defect in viral DNA replication, qPCR was performed to measure the levels of viral DNA synthesis in vAc111KO-transfected cells during a single replication cycle. vAcWT was used as a positive control. Total cellular DNA was isolated from vAc111KO-or vAcWT-transfected cells at the designated time points, and treated with DpnI to eliminate all input bacmid DNA. As shown in Figure 4, viral DNA replication levels of vAc111KOtransfected cells and vAcWT-transfected cells were comparable throughout a 24-h time course. The qPCR results demonstrated that the deletion of ac111 did not affect the onset or levels of viral DNA synthesis.

Ac111 is Not Required for Viral DNA Replication
To assess whether ac111 deletion leads to a defect in viral DNA replication, qPCR was performed to measure the levels of viral DNA synthesis in vAc111KO-transfected cells during a single replication cycle. vAcWT was used as a positive control. Total cellular DNA was isolated from vAc111KO-or vAcWT-transfected cells at the designated time points, and treated with DpnI to eliminate all input bacmid DNA. As shown in Figure 4, viral DNA replication levels of vAc111KO-transfected cells and vAcWT-transfected cells were comparable throughout a 24-h time course. The qPCR results demonstrated that the deletion of ac111 did not affect the onset or levels of viral DNA synthesis.

Ac111 is Not Essential for Virus Morphogenesis
Electron microscopy observations were performed using thin sections of vAc111KO-, vAcWT-, and vAc111:HA-transfected Sf9 cells at 72 h p.t., to further investigate whether the ac111 deletion had any effect on virus morphogenesis ( Figure 5). In vAc111:HA-transfected Sf9 cells, the typical characteristics were similar to those of vAcWT-transfected cells, including a well-defined virogenic

Ac111 is Not Essential for Virus Morphogenesis
Electron microscopy observations were performed using thin sections of vAc111KO-, vAcWT-, and vAc111:HA-transfected Sf9 cells at 72 h p.t., to further investigate whether the ac111 deletion had any effect on virus morphogenesis ( Figure 5). In vAc111:HA-transfected Sf9 cells, the typical characteristics were similar to those of vAcWT-transfected cells, including a well-defined virogenic stroma (VS) enriched with electron-dense, rod-shaped nucleocapsids in the center of the nucleus ( Figure 5A), and developing OBs embedding numerous mature ODVs in the ring zone ( Figure 5B). The cells transfected with vAc111KO exhibited morphologically indistinguishable characteristics to those of vAc111:HA and vAcWT ( Figure 5C,D). These results indicated that the deletion of ac111 did not affect virus morphogenesis.

Ac111 Plays a Role in Oral Infectivity in a Host-Dependent Manner
To determine the effect of ac111 deletion on the infectivity of AcMNPV in insect larvae, bioassays with BVs and OBs grown in Sf9 cells were performed in two host species, T. ni and S. exigua. Intrahemocoelic injection of vAc111KO BV supernatant into fourth-instar T. ni at 1 TCID50 units/larva produced 67% mortality, which was not significantly different from that of vAc111:HA (63%) or vAcWT (63%) (Student's t-test, p> 0.05) ( Table 2). Intrahemocoelic injection of vAc111KO, vAc111:HA

Ac111 Plays a Role in Oral Infectivity in a Host-Dependent Manner
To determine the effect of ac111 deletion on the infectivity of AcMNPV in insect larvae, bioassays with BVs and OBs grown in Sf9 cells were performed in two host species, T. ni and S. exigua. Intrahemocoelic injection of vAc111KO BV supernatant into fourth-instar T. ni at 1 TCID 50 units/larva produced 67% mortality, which was not significantly different from that of vAc111:HA (63%) or vAcWT (63%) (Student's t-test, p> 0.05) ( Table 2). Intrahemocoelic injection of vAc111KO, vAc111:HA or vAcWT BV supernatant into fourth-instar S. exigua at 2 TCID 50 units/larva produced 70%, 67%, or 70% mortalities, respectively, and there was no significant difference among the mortalities (Student's t-test, p> 0.05) ( Table 2). However, bioassays with OBs revealed that ac111 affected the oral infectivity of AcMNPV in the two host species in a different way. Bioassays with OBs in T. ni larvae showed that, the LD 50 values of vAc111:HA and vAcWT were 59 and 54 OBs/larva, respectively with overlapping 95% confidence interval indicating they were not significantly different, the LD 50 of vAc111KO (300 OBs/larva) was approximately five times more than that of vAcWT ( Table 2). The LT 50 value of vAc111:HA (86 h) was comparable to that of vAcWT (85 h), but the LT 50 value of vAc111KO (106 h) was 21 h longer than that of vAcWT (Table 2). Therefore, the deletion of ac111 reduced the infectivity and killing speed of AcMNPV in T. ni larvae. However, in S. exigua larvae, no significant differences in infectivity or killing speed were observed among vAc111KO, vAc111:HA and vAcWT, with the LD 50 values being 5006, 4650, and 4535 OBs/larva, and the LT 50 values being 148 h, 141 h and 140 h, respectively (as determined by overlapping 95% confidence interval) ( Table 2). This suggests that the deletion of ac111 did not impact per os infectivity of AcMNPV in S. exigua larvae.
Taken together, the bioassays demonstrated that ac111 plays a role in primary oral infection of AcMNPV and the effect is host dependent.

Discussion
ac111 is one of the highly conserved lepidopteran-specific baculovirus genes, but its role in the AcMNPV replication remains unknown. In this study, we demonstrated that ac111 is important for AcMNPV to efficiently establish per os infection in T. ni larvae specifically, while is dispensable for per os infectivity of AcMNPV in S. exigua larvae, thus providing evidence for its important role as a conserved baculovirus gene.
To investigate the function of ac111 in the AcMNPV life cycle, an ac111-knockout virus was generated and characterized. We found that ac111 was dispensable for virus replication in vitro as evidenced by indistinguishable characteristics such as a normal viral growth curve, normal plaque, steady viral DNA replication and normal virus morphogenesis, compared to those of vAcWT. These results were consistent with the study on Bm93 [33]. Bioassays showed that there was no significant difference between the LD 50 and LT 50 of vAc111KO OBs and those of vAcWT in S. exigua larvae. However, the LD 50 of vAc111KO OBs was approximate five times higher than that of vAcWT OBs in T. ni larvae, and the LT 50 of vAc111KO was 21 h longer than that of vAcWT in T. ni larvae. Furthermore, the deletion of ac111 did not affect BV infectivity through intrahemocoelic injection in T. ni and S. exigua larvae. Taken together, these results indicated that ac111 affects the per os infectivity of AcMNPV in a host-dependent manner.
Of the 154 putative ORFs encoded in the AcMNPV genome, the function of more than 70% of these ORFs has been determined. While some genes were found to be nonessential and could be deleted without affecting virus replication, some genes are essential for virus replication in vitro and participate in critical steps in the virus life cycle, such as DNA replication, early/late gene transcription, or nucleocapsid assembly. There are some other genes that are dispensable for virus replication in vitro but function during oral infection of insect larvae. Genes belonging to this category include the nine pifs and several virus virulence-related genes, such as ac111, ac145, and ac150. Baculoviruses initiate infection orally when their insect hosts consume and digest OBs in the alkaline pH of the midgut, releasing ODVs to infect the midgut epithelial cells. A number of viral membrane proteins, including the PIF proteins, have been identified to be essential for this process. Considering the pivotal role of PIFs, these proteins are encoded by baculovirus core genes [6], indicating that they play pivotal roles in per os infection of all baculoviruses and thus have been conserved during baculovirus evolution. Other virus virulence-related genes are only conserved in certain baculoviruses, implying that they might function distinctly compared to PIFs in a host dependent manner and facilitate the efficient establishment of baculovirus infection in specific hosts. Similar to ac111, previous studies have demonstrated that two AcMNPV genes, ac145 and ac150 affect oral infectivity in a host dependent manner [26]. ac145 and ac150 encode proteins related to the 11K proteins with a characteristic C 6 motif of conserved cysteine residues in a well-defined spacing pattern. Previous studies have found that the deletion of ac145 led to a six-fold drop in infectivity of AcMNPV in T. ni larvae but not in Heliothis virescens larvae [26]. On the other hand, the deletion of ac150 had no effect on infectivity of AcMNPV in either host [26]. However, the deletion of both ac145 and ac150 significantly reduced the infectivity of AcMNPV (39-fold) in H. virescens larvae [26]. In another study, significantly more OBs were required per LD 50 for ac150 deletion AcMNPVcompared with wild-type virus in H. virescens, S. exigua, and T. ni larvae (4.1-, 5.6-, and 18-fold increases, respectively), suggesting that Ac150 indeed had an enhancing effect on per os infection and that the effect was host-specific [31]. In this study, we showed that ac111 specifically affected viral infectivity and killing speed of AcMNPV in T. ni larvae but is dispensable in S. exigua larvae. Therefore, although AcMNPV could establish infection in T. ni, S. exigua, and H. virescens larvae, differences might exist that subtly affect the fitness of AcMNPV in these different insect hosts. It is intriguing to speculate that a group of proteins in AcMNPV, such as Ac111, Ac145, and Ac150, might specifically affect the infectivity of a virus in different hosts. We also cannot rule out the possibility that these viral proteins might interact with each other or with specific host proteins, and these interactions may eventually determine the host tropism of AcMNPV. Further studies are needed to verify this assumption.
Taken together, this study has shown that ac111 was not required for AcMNPV replication in vitro, but it played a pivotal role in the viral infectivity in vivo in T. ni but not in S. exigua larvae. Further experiments are needed to unravel the specific role of ac111 during AcMNPV infection in T. ni larvae. These studies will likely shed light on our understanding of the mechanism of baculovirus-insect interactions, to ascertain the fitness of baculoviruses in different hosts.