Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus identified two new occlusion-derived virus-associated proteins, HA44 and HA100

Abstract

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.

FIG. 1.

SDS-PAGE profile and MS results of purified HearNPV ODV. ODV proteins were separated by 12% SDS-PAGE and stained with colloidal blue. The ODV bands (numbered in the middle) were subjected to MALDI-TOF MS, and their identities are listed on the right.

FIG. 2.

Alignment of the amino acid sequences of HA44 and its homologues among the group II NPVs. Three shading levels were set, black for 100% identity, dark gray for 80% identity, and light gray for 60% identity. The NCBI accession numbers are NP_818692 for AdhoNPV45, YP_529786 for ORF116 of Agrotis segetum NPV (AgseNPV116), YP_249646 for ORF42 of Chrysodeixis chalcites NPV (ChchNPV42), NP_075113 for HA44, NP_542668 for ORF45 of HzSNPV45, NP_047691 for ORF55 of Lymantria dispar NPV (LdMNPV55), NP_613219 for ORF136 of Mamestra configurata NPV A (MacoA136), NP_689309 for ORF135 of Mamestra configurata NPV B (MacoB135), NP_037867 for ORF107 of Spodoptera exigua NPV (SeMNPV107), NP_258314 for ORF44 of Spodoptera litura NPV (SpliNPV44), and YP_308929 for ORF39 of Trichoplusia ni NPV (TnSNPV39).

FIG. 3.

Neighbor-joining tree derived from HA44 and its homologues among NPVs and GVs. Bootstrap values (1,000 replicates, nodes supported with more than 50%) are on the branch lines. The accession numbers for NPVs are as described in the legend to Fig. 3. The additional ones are NP_872567 for ORF113 of Adoxophyes orana GV (AdorGV113), YP_006220 for ORF124 of Agrotis segetum GV (AgseGV124), NP_891969 for ORF122 of Cryptophlebia leucotreta GV (CrleGV122), NP_148919 for ORF135 of Cydia pomonella GV (CpGV135), NP_663288 for ORF123 of Phthorimaea operculella GV (PhopGV123), NP_068332 for ORF113 of Plutella xylostella GV (PlxyGV113), and NP_059320 for ORF172 of Xestia c-nigrum GV (XecnGV172).

FIG. 4.

Comparison of PARGs from a wide range of organisms and from group II NPVs. A, putative regulatory domain; B, C, and D, catalytic fragments; C, PARG catalytic domain. Percent conservation is indicated in each block with respect to the bovine PARG. The amino acid positions of bovine PARG domains and the lengths of PARGs are indicated. The accession numbers of PARGs are NP_776563 for Bos taurus, AAH52966 for Homo sapiens, NP_036090 for Mus musculus, NP_112629 for Rattus norvegicus, NP_477321 for Drosophila melanogaster, NP_501508 for Caenorhabditis elegans, NP_075169 for HA100, NP_818756 for AdhoNPV, YP_529728 for AgseNPV, YP_249712 for ChchNPV, NP_542726 for HzSNPV, NP_047778 for LdMNPV, NP_613153 for MacoNPV A, NP_689244 for MacoNPV B, NP_037812 for SeMNPV, NP_258370 for SpliNPV, and YP_308992 for TnSNPV.

FIG. 5.

Western blot analysis of the HearNPV ODV/BV nucleocapsid (NC) and envelope (E) fractions with anti-HA44 and anti-HA100 antibodies. Healthy HzAM1 cells (H) and virus-infected cells (I) were loaded as negative and positive controls. VP80, ODV-E56, and the F protein were detected by their specific antibodies for illustrating the NC-specific protein, ODV E-specific protein, and BV E-specific protein, respectively. M, molecular size markers.

FIG. 6.

IEM of HA44 and HA100 and localization in HearNPV ODVs and nucleocapsids (NC) of ODVs. A 1:100 dilution of anti-HA44 or anti-HA100 antiserum was used as the primary antibody. Preimmune serum was used as a negative control. A, IEM of HA44; B, IEM of HA100. Bars, 100 nm.