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Baculoviruses are rod-shaped, enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 kb to 180 kb. During their life cycle, baculoviruses produce two distinct phenotypes: the budded virus (BV), which is responsible for the spread of virus from cell to cell within and the occlusion-derived virus (ODV), which is responsible for the horizontal transmission of infection between insects [4].
The infection of baculovirus is accompanied with polymerization of granular actin (G-actin) to filamentous actin (F-actin) in the host nucleus, where viral nucleocapsids are assembled and F-actin is supposed to facilitate this process [3, 10]. The actin polymerization process is mediated by the interaction between the WCA domain of N-WASP and the actin-related protein 2/3 (Arp2/3) complex [6, 7]. Additionally, the phosphorylation of N-WASP at 256Tyr by Abl kinases has been shown to be critical for the actin comet tail formation [1].
Previously, we had demonstrated that HearNPV HA2, a viral nucleocapsid protein bearing a WCA domain is essential for baculovirus-induced host nuclear actin polymerization [8, 12]. Also, knockout of ha2 from the viral genome is lethal to HearNPV, as manifested by lack of nuclear F-actin formation and aberrant viral nucleocapsid formation in the nucleus of virus transfected cells [13].
Since the phosphorylation status of N-WASP is correlated with its capability in initiating actin polymerization, and HA2 counterpart P78/83 of AcMNPV is a phosphorylated protein [9], the function of putative phosphorylation sites of HA2 was investigated with respect to viral replication and actin polymerization.
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Insect cell line HzAM1 was maintained in Grace's medium (Invitrogen) with a supplement of 10% fetal bovine serum (FBS) (Invitrogen) at 27℃. All the HearNPV recombinant bacmid constructs were derived from HabacHZ8 and propagated in Escherichia coli strain DH10B [11].
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HA2 sequence was submitted to either Prosite (http://www.expasy.org/tools/scanprosite/) to identify potential phosphorylation sites, or NCBI Blast (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) to make sequence alignments and for motif recognition.
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The bacmid with ha2 knockout (HaΔha2) was previously described [13]. The site-directed mutagenesis of ha2 was performed using a two-step polymerase chain reaction (PCR), following the protocols described previously [14]. Briefly, ha2 was cloned to pFbdg using primer set Ha2-upper/lower (Table 1) [14]. To introduce mutations to ha2, primer sets 232-upper/lower, 245-upper/lower, and 250-upper/lower (Table 1) were used to generate donor plasmids bearing T232A, S245G, and S250A, respectively. The resulted plasmids were transposed to HaΔha2 by using the Bac-to-Bac method [5, 11, 13], and generated vHaha2-T232A, vHaha2-T245G, and vHaha2-S250A (Fig. 1A). The resulted bacmid constructs were confirmed by PCR with primer set M13+/M13-, according to the Bac-to-Bac protocol (Invitrogen).
Table 1. Primers used in this research
Figure 1. Construction of the recombinant bacmids. A: Diagram of bacmid constructs. Coding sequences of EGFP and HA2 mutants were respectively cloned into donor plasmid pFastBac-dual. The resulted plasmids were transposed to HaΔha2 to generate recombinant bacmid constructs vHaha2-T232A, vHaha2-S245G, and vHaha2-S250A. B: Confirmation of the recombinant bacmids by PCR with M13± primers.
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These bacmid constructs were transfected to HzAM1 cells using lipofectin (Invitrogen). At 144 hours post transfection (hpt), the supernatants were collected and filtered through 0.45 µm-diameter syringe filters (Sartorius) to remove cell debris before being added to fresh HzAM1 cells to initiate secondary infection. After 60 min of incubation, the supernatant was discarded and replenished with fresh Grace medium plus 10% FBS. At 144 hours post infection (hpi), the cell images were captured with an Olympus-IX51 microscope.
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Cell transfection was achieved using a virus replication assay. At 59 hpt or 96 hpt, the transfected cells were rinsed twice with 1× phosphate buffered saline (PBS) (Beyotime), fixed in 4% paraformaldehyde (Sigma), and penetrated in 0.2% Triton-X (Sigma). The cells were then stained with rhodamine-conjugated phalloidin (Invitrogen) and Hoechst 33258 (Beyotime) for labeling of F-actin and nuclear DNA, respectively. The images were captured using a Leica SP2 confocal laser scanning microscope.