WSSV has bacilliform, enveloped, non-occluded virions containing a double stranded (ds)DNA genome. The virus has a wide host range, where more than 93 species of arthropods have been reported as hosts or carriers of WSSV. The mechanism of WSSV infection and propagation in the host cell remains unknown in spite of its severe impact on the shrimp farming and that understanding of the virus-host interaction is likely to be the key point in developing strategies to prevention of this disease outbreak.
The invasion of WSSV into the penaeid shrimps affects their immune defense responses. The molecular changes associated at the gene transcript and protein expression levels in the shrimp immune system have been investigated using expressed sequenced tag (EST), DNA microarray and proteomic analyses. The, up-regulated gene transcripts or proteins have been further characterized for their potential role in both the cellular and humoral immunity (defense responses) of shrimps in response to WSSV infection. These were found to include the antimicrobial peptides, prophenol oxidase (proPO) system, oxidative stress, proteinases and proteinase inhibitors. Moreover, three of the major immune responses (phagocytosis, apoptosis and the proPO cascade) have been compared to study their role in the antiviral defense system.
The novel proteins that are up-regulated in shrimps following WSSV infection are typically viewed as interesting molecules to characterize their function in the shrimp immune system. For example, the novel viral responsive protein, hemocyte homeostasis-associated protein (HHAP), was found to be highly up-regulated at both the transcript and protein levels in WSSV-infected shrimp hemocytes. Silencing of this gene in Penaeus monodon (PmHHAP) by dsRNA-interference (RNAi) caused damage to shrimp hemocytes and a severe decrease in their numbers, suggesting the important role of PmHHAP in hemocyte homeostasis. Suppression subtractive hybridization (SSH) and microarray analyses (our unpublished data) of WSSV-challenged P. monodon hemocytes identified the novel viral responsive protein (VRP) PmVRP15 as one of the most highly up-regulated genes in the acute phase of WSSV-infected hemocytes. Herein, we attempt to characterize the function of PmVRP15 from P. monodon by RNAi-mediated gene silencing. Fluorescence-labeling along with confocal laser scanning microscopy (CLSM) was used to examine the localization of PmVRP15 in shrimp hemocytes. Overall, the likely importance of this novel protein in promoting viral propagation was suggested.
Results
The full-length cDNA of PmVRP15 and sequence analysis
A partial sequence of the PmVRP15 cDNA was initially obtained from the SSH library of WSSV-challenged P. monodon hemocytes. The full-length cDNA of PmVRP15 was then obtained using 5′ RACE (GenBank accession code KF683338), and was found to contain 722 base pairs with a deduced complete open reading frame encoding for a predicted 137 amino acids whose predicted molecular mass of 15.036 kDa (Fig. 1). The size of the deduced PmVRP15 cDNA was confirmed by Northern blot analysis where the detected mRNA had a corresponding size of about 722 base pairs (data not shown). The BLAST homology search of the GenBank database using blastP program indicated that the putative predicted protein sequence of PmVRP15 has the highest similarity to a hypothetical protein AGAP000432-PA (XP_310667.1) from mosquito Anopheles gambiae with a significant E value of 2e−11, 35 per cent identity and 58 per cent similarity. Lower significant similarity of PmVRP15 with other five hypothetical proteins such as conserved hypothetical protein (XP_001849829.1) from Culex quinquefasciatus, GE16519 (XP_002099810.1) from Drosophila yakuba, GK10098 (XP_002071654.1) from Drosophila willistoni, hypothetical protein AaeL_AAEL014657 (XP_001649261.1) from Aedes aegypti, and PREDICTED protein C19orf12 homolog (XP_004536446.1) from Ceratitis capitata, was also found with the E value range from 10−8–10−5. Protein-structural analysis revealed a likely transmembrane helix of 23 amino acids (TMHMM Server v. 2.0, available on-line) [27] but with no predicted signaling domain (Simple Modular Architecture Research Tool (SMART), available on-line).
PmVRP15 gene expression in P. monodon tissues
The tissue distribution of PmVRP15 transcripts in normal shrimps was examined by RT-PCR, where PmVRP15 transcripts were found in all tested tissues but was highly expressed in hemocytes followed by lymphoid tissue and then with moderate to low levels in the heart, gill, hepatopancreas and intestine, and low levels in the antennal gland, epipodite and eye stalk (Fig. 2).
Up-regulation of PmVRP15 in response to WSSV infection in P. monodon hemocytes
Our previous results from SSH and microarray analyses (unpublished data) of WSSV-challenged P. monodon hemocytes revealed that PmVRP15 is one of the most highly up-regulated genes in the acute phase of WSSV-infected hemocytes. Herein, PmVRP15 transcript levels in P. monodon hemocytes were evaluated by qRT-PCR. The results clearly confirmed that PmVRP15 transcripts were highly up-regulated in the shrimp hemocytes after WSSV challenge, increasing by about 3.6-, 9410- and 1351-fold at 24, 48 and 72 hpi, respectively, compared to that in unchallenged shrimp hemocytes (Fig. 3). Furthermore, the PmVRP15 protein level was up-regulated in WSSV-infected shrimp hemocytes, as determined by Western blot analysis using the polyclonal rabbit anti-rPmVRP15 antibody (Fig. 4), where the detected 15 kDa protein band corresponded to the predicted size of PmVRP15 protein. These results are consistent with a role for PmVRP15 in response to WSSV infection.
Localization of PmVRP15 and VP28 in uninfected and WSSV-infected P. monodon hemocytes
The location of PmVRP15 and VP28 proteins in hemocytes and the potential cell type(s) that produce the protein was examined by CLSM using the antibodies specific to PmVRP15 and the WSSV late protein VP28 coupled with different fluorescence-conjugated secondary antibodies. Since PmVRP15 and VP28 were detected as green and red fluorescence, respectively, the accepted fraction of the emission spectra of TO-PRO-3, used to stain the nuclear DNA, was adjusted to show as blue. Three types of hemocytes (hyaline, semigranular and granular cells) were visible in the bright field image (Fig. 5A). In the uninfected (control) shrimp hemocytes, all three types of hemocytes were weakly positive for PmVRP15, and the protein was localized in the cytoplasm near to the nuclear membrane (Fig. 5B). In the WSSV-infected shrimps, the PmVRP15 protein expression level was hardly detected at 6 hpi but significantly up-regulated at 24 hpi (not shown) and 48 hpi (Fig. 5) and in the moribund shrimps (not shown). Interestingly, PmVRP15 and VP28 protein expression were found in the same hemocytes at the late infection phase (48 hpi) and the moribund stage of viral infection (Fig. 5 and not shown, respectively). Thus, the expression of PmVRP15 in P. monodon hemocytes appears to be linked to a response to the acute phase of WSSV infection.
Effect of PmVRP15 gene knockdown on viral propagation in P. monodon hemocytes
Since PmVRP15 transcripts and protein were found to be highly up-regulated in the hemocytes of WSSV-infected shrimps, then the potential importance of PmVRP15 in the shrimp's response to WSSV infection was evaluated using RNAi-mediated gene knockdown by injection of PmVRP15 dsRNA. Injection of dsRNA PmVRP15 specifically suppressed the PmVRP15 transcription levels in shrimp hemocytes at 24 hpi whereas the injection with GFP dsRNA had no effect on PmVRP15 mRNA expression levels (Fig. 6A). The suppression of PmVRP15 expression at the translational level was also confirmed. Twenty-four hour after knocking-down PmVRP15 gene in WSSV-infected shrimp, PmVRP15 protein expression level in the shrimp hemocyte lysate was compared with that of the control WSSV-infected shrimp with GFP dsRNA injection. The result showed that PmVRP15 protein expression level in WSSV-challenge shrimp was significantly decreased after PmVRP15 gene silencing (Fig. 6B).
The transcript expression level of representative WSSV genes for the three stages of WSSV infection; namely ie-1 (very early stage), wsv477 (early stage) and vp28 (late stage), was determined after PmVRP15 knockdown in WSSV-infected shrimp by qRT-PCR. The transcript expression level of all three viral genes tested was considerably decreased in the PmVRP15 knockdown shrimps (by 83.5 per cent, 85.5 per cent and 94.8 per cent for ie-1, wsv477 and vp28, respectively) compared to the control shrimps (Fig. 7). The decrease in WSSV transcript levels suggested that PmVRP15 might participate in the WSSV propagation process.
Cumulative mortality of P. monodon shrimp after PmVRP15 gene knockdown
As stated above, after PmVRP15 gene knockdown in WSSV-infected shrimp, the expression level of representative WSSV genes was significantly decreased suggesting the involvement of PmVRP15 in the WSSV propagation. PmVRP15 gene was silenced in WSSV-infected P. monodon and the mortality of shrimp was observed in parallel to those silenced with GFP dsRNA. The cumulative mortality result showed that, after 66–102 hours post-WSSV infection, mortality rate of PmVRP15 knockdown group was 50 per cent lower than that of control group (The shrimp mortality reached 100 per cent at 90 hpi) (Fig. 8A). However, after 102 hpi, the cumulative mortality of PmVRP15 knockdown shrimp was gradually increased and reached 100 per cent at 144 hpi (6 dpi). Due to the fact that PmVRP15 gene is highly up-regulated after WSSV infection, here, the PmVRP15 gene recovery after PmVRP15 dsRNA and WSSV injection was determined. Figure 8B showed that PmVRP15 gene was recovered for about 50 per cent at 36 hpi and to the same level as in the control at 60 hpi. According to the results, we confirmed that the absence of PmVRP15 gene in shrimp affected the mortality of WSSV-infected shrimp.
Discussion
To study the mechanism of WSSV infection and propagation, an understanding of the immune response of shrimps is an important key. PmVRP15 transcripts were found in all tissues examined of uninfected P. monodon shrimps, but were mainly expressed in the hemocytes. Hemocytes are the major immune cells of shrimps and play an essential role in both the cellular and humoral immune responses. Three different types of hemocytes (granular, semigranular and hyaline cells) have been classified in shrimp hemolymph. In crustaceans, specific (but partially overlapping) functions have been attributed to the different hemocyte types, such as phagocytosis in hyaline cells, encapsulation, phagocytosis, ProPO system and cytotoxicity in semigranular cells, and the ProPO system and cytotoxicity in granular cells. In contrast, the PmVRP15 protein was located in all three types of hemocytes, suggesting that PmVRP15 may have a more constitutive or broad immune based function. Upon WSSV infection, the expression of PmVRP15 transcripts and protein were both up-regulated in P. monodon hemocytes, exclusively within WSSV-infected ones.
From the SSH analysis (our unpublished data), PmVRP15 transcripts appeared to be highly expressed in the acute phase of WSSV-infected P. monodon hemocyte; however, the function of its gene product have not been characterized. Interestingly, several hemocyte proteins were found to be significantly altered in their expression levels in the different stages of virus infection, including both well-characterized proteins and those of currently unknown function. Several cognate immunity proteins involved in viral defense responses have been found to be up-regulated in the early phase of viral infection, such as the antimicrobial peptides ALFPm3, Peneidin5 and hemocyanin. During the acute phase of WSSV infection, the host immune responses and mechanism(s) used are not yet fully understood, but several host proteins have previously been identified that show altered expression levels, including the scavenger receptor and transglutaminase amongst others. Recently, the unknown function PmHHAP, which is highly up-regulated in viral infected shrimps, was identified and characterized as a novel responsive protein that plays an important role in hemocyte homeostasis.
Nuclear membrane proteins have been reported in many vertebrates to act as a path of infection for viruses, such as influenza virus and herpes virus. However, such protein functions remain unknown in invertebrates. Herein, we found that the expression of PmVRP15 was mainly located at the nuclear membrane of P. monodon hemocytes. Moreover, hemocytes that were infected with WSSV also expressed PmVRP15 at high levels. It would be interesting to study how a severe WSSV infection can stimulate PmVRP15 expression. In addition, the data presented here may represent the first report linking a correlative relationship between a potential P. monodon nuclear membrane protein (PmVRP15) and WSSV infection. However, an actual direct causative role, and the mechanism of such, remains to yet be established.
In the acute viral infection phase, the host cells not only express defensive molecules that play a role in protecting the host cell against the virus, but the virus uses the host machinery to express viral proteins for propagation, including the immediate early, early and late genes. At this stage the host cell loses the ability to regulate gene expression and is seconded to perform virus multiplication. Although cell death by apoptosis is one last line of host defense, whereby the infected cell is self-signaled for destruction to prevent viral replication and so to protect against viral spread to other cells, some viral proteins can inhibit the apoptosis system, including in WSSV the anti-apoptosis protein-1, AAP-1 and WSSV222. The high expression level of PmVRP15 found here in WSSV-infected P. monodon hemocytes is in agreement with (but not conclusive for) that PmVRP15 is up-regulated to mediate viral propagation in the acute phase of infection, since PmVRP15 gene knockdown resulted in a significant decrease in viral gene expression, as observed for ie-1 (an immediate early gene), wsv477 (an early gene) and vp28 (a late gene) transcripts and in the delay of shrimp death upon WSSV infection. Additionally, PmVRP15 protein was found to be localized near the nuclear membrane in the cytoplasm of WSSV-infected hemocytes which coupled with the predicted presence of transmembrane domain, suggests it may function at least in part as a nuclear membrane (or proximally related membrane) protein. If so, this is in accord with the notion that the host machinery was used to transport the viral components in the host cell, as found in the transmembrane protein PmRab7. The interaction of viral and host proteins is a potentially important key to answer the function of PmVRP15 in WSSV-infected cells, and could initially be addressed by, for example, using co-immunoprecipitation or the yeast two-hybrid screening assays. Nevertheless, the mechanism of control of expression (transcriptional control) of the PmVRP15 gene would also be interesting to elucidate, including characterization of the promoter. These aspects are now under investigation in an attempt to reveal the mechanism and regulation of PmVRP15 in WSSV propagation in P. monodon hemocytes.
Conclusion
The cDNA of a novel viral responsive gene from the black tiger shrimp (P. monodon), PmVRP15, was cloned and sequenced to acquire the full-length cDNA coding sequence. Expression analysis showed PmVRP15 transcripts were mainly found in hemocytes and along with the PmVRP15 protein were highly up-regulated in WSSV-infected hemocytes. PmVRP15 protein was localized at or near the nuclear membrane of uninfected and WSSV-infected shrimp hemocytes. After RNAi-mediated PmVRP15 suppression, WSSV propagation and shrimp mortality were markedly decreased. The function of PmVRP15 is unknown but it possibly plays a role in WSSV propagation in shrimp hemocyte.
April 2014
