Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin

Abstract

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected with AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released.

Figure 1. Cloning and expression of TnGlv1 and TnGlv2

(A) Diagram of TnGlv1 (top) and TnGlv2 (bottom) with the N-terminal signal peptide, putative transmembrane domains noted, and locations of C-terminal 6x-His and V5 epitope tags. (B) PCR analysis of the cloned TnGlv1 and TnGlv2 inserted into expression vectors. Bands of the predicted molecular weights were observed for TnGlv1 and TnGlv2 inserted in top ENTR-TnGlv1 cl12 and pENTR-TnGlv2 cl10 TA vectors (702 bp and 665 bp, respectively) using PCR with M13 forward and reverse primers. (C) Western blot of TnGlv1 and TnGlv2 protein present in the supernatants of Sf9 cells stably transfected with gloverin expression vectors (TnGlv1 Sup, TnGlv2 Sup) and of Ni-NTA affinity column purified gloverin protein (TnGlv1 Ni-NTA and TnGlv2 Ni-NTA; 60 s exposure).

Figure 2. TnGlv1 and TnGlv2 reduced the quantity of infectious AcMNPV-hsp70/lacZ BV

(A) Infectious BV was reduced when incubated in the presence of Sf9 cell culture supernatants containing TnGlv1 (TnGlv1 Sup), TnGlv2 (TnGlv2 Sup) or Ni-NTA affinity column purified TnGlv1 (TnGlv1 Ni-NTA) or TnGlv2 (TnGlv2 Ni-NTA). The results from replicated plaque assay experiments were normalized to the vehicle treatment (TNM-FH media) for each experiment, which was set to 100 % infectious BV, and the change in the quantity of infectious BV reported as the percent of the vehicle treatment. Means and SEM were calculated from N ≥ 3 replicates per treatment. (B) Plaque assay of Sf9 cells showing blue LACZ-positive plaques of AcMNPV-hsp70/lacZ BV incubated with cell culture supernatants containing TnGlv1, TnGlv2, or vehicle. (C) Concentration dependent antiviral activity of TnGlv1 and TnGlv2 on AcMNPV BV. Plaque assays of AcMNPV-hsp70/lacZ BV incubated with increasing concentrations of affinity column purified TnGlv1 and TnGlv2 protein was correlated with decreasing quantities of infectious BV relative to vehicle treatments (BV incubated with TNM-FH media; N = 4 replicates per treatment).

Figure 3. Gloverin-mediated release of calcein from phosphatidylglycerol (PG) or phosphatidylcholine (PC) vesicles

(A) Unilamellar vesicles comprised of PG and encapsulated with calcein were incubated with nanomolar concentrations of affinity purified TnGlv1 or TnGlv2 and the quantity of calcein released quantified using a fluorescence spectrophotometer (N = 3 replicates per concentration and treatment). Increasing concentrations of TnGlv1 or TnGlv2 protein resulted in concomitant increases in calcein released from the PG vesicles. (B and C) Individual trials showing the release of calcein over time from PG vesicles incubated with multiple concentrations of TnGlv1 (B) or TnGlv2 (C). Maximum gloverin-mediated calcein release from PG vesicles occurred within 2 min of the gloverin addition to the reaction cuvette. (D and E) TnGlv1 and TnGlv2 (100 nM) did not cause calcein release from PC vesicles after an 8 min incubation period. Solid arrows indicate the time that test proteins were added to the reaction cuvettes and broken arrows indicate the time that detergent was added to each reaction cuvette to cause full release of the encapsulated calcein from the vesicles.

Figure 3. Gloverin-mediated release of calcein from phosphatidylglycerol (PG) or phosphatidylcholine (PC) vesicles

(A) Unilamellar vesicles comprised of PG and encapsulated with calcein were incubated with nanomolar concentrations of affinity purified TnGlv1 or TnGlv2 and the quantity of calcein released quantified using a fluorescence spectrophotometer (N = 3 replicates per concentration and treatment). Increasing concentrations of TnGlv1 or TnGlv2 protein resulted in concomitant increases in calcein released from the PG vesicles. (B and C) Individual trials showing the release of calcein over time from PG vesicles incubated with multiple concentrations of TnGlv1 (B) or TnGlv2 (C). Maximum gloverin-mediated calcein release from PG vesicles occurred within 2 min of the gloverin addition to the reaction cuvette. (D and E) TnGlv1 and TnGlv2 (100 nM) did not cause calcein release from PC vesicles after an 8 min incubation period. Solid arrows indicate the time that test proteins were added to the reaction cuvettes and broken arrows indicate the time that detergent was added to each reaction cuvette to cause full release of the encapsulated calcein from the vesicles.

Figure 4. Transcriptional analysis of gloverin in hemocytes isolated from AcMNPV-infected T. ni larva

Gene expression was analyzed using semi-quantitative RT-PCR for hemocytes isolated from T. ni 12 h after inoculation with AcMNPV-hsp70/lacZ BV or vehicle (TNM-FH). Relative to GAPDH expression, TnGlv2 was upregulated 5.7-fold.

Figure 5. Hypothetical mechanisms of action for the observed antiviral activities of gloverin

Top panels. GP64 is the attachment and fusion protein of AcMNPV BV. The overall negative charge of GP64 or clusters of negatively charged amino acids localized near the transmembrane domain of GP64 may attract the positively charged amino acids of gloverin to the surface of BV. Gloverin may subsequently hinder the conformational changes of GP64 needed to initiate an infection. Bottom panels. Gloverin may interact with negatively charged amino acids of other proteins localized to the envelope or with negatively charged phospholipids of the envelope membrane. Accumulated gloverin on the surface of BV may cause membrane strain or pores to form that disrupt the BV envelope. Illustration credit: Emily L. Ling.