Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import,nucleolar localization and nuclear export signal sequences

The order Nidovirales, which includes the arteriviruses and coronaviruses, incorporate a cytoplasmic replication scheme; however, the nucleocapsid (N) protein of several members of this group localizes to the nucleolus suggesting that viral proteins influence nuclear processes during replication. The relatively small, 123 amino acid, N protein of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, presents an ideal model system for investigating the properties and mechanism of N protein nucleolar localization. The PRRSV N protein is found in both cytoplasmic and nucleolar compartments during infection and after transfection of gene constructs that express N-enhanced green fluorescent protein (EGFP) fusion proteins. Experiments using oligopeptides, truncated polypeptides and amino acid-substituted proteins have identified several domains within PRRSV N protein that participate in nucleo-cytoplasmic shuttling, including a cryptic nuclear localization signal (NLS) called NLS-1, a functional NLS (NLS-2), a nucleolar localization sequence (NoLS), as well as a possible nuclear export signal (NES). The purpose of this paper is to review our current understanding of PRRSV N protein shuttling and propose a shuttling scheme regulated by RNA binding and post-translational modification.     

Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus

A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.     

Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27

Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to ⁸¹RRAR⁸⁴ and ⁸⁶RPRRPRRRPRRR⁹⁷ respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS + NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS + NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus.     

Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha

Betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) protein alpha, a virus capsid protein, was detected in both nucleolus and cytoplasm of infected cells of Asian sea bass (SB) and transfected cells of SB and Cos-7 with pcDNA3.1/RNA2. To study its subcellular localization, ORF of protein alpha with 338 aa was fused with enhanced green fluorescent protein (EGFP) gene and was detected in transfected cells in the absence of other viral proteins. In both SB and Cos-7 cells, protein alpha was found to localize EGFP to the nucleolus and cytoplasm. Deletion mutants of protein alpha indicated that N-terminal 43 amino acid residues were required to import EGFP-alpha protein into the nucleolus. Further deletions within the 43 amino acid backbone, EGFP/33aa(1-33) and EGFP/30aa(14-43), localized to the nucleolus, suggesting that the 20 amino acids from 14 to 33 of protein alpha were the domain of nucleolus localization. To further determine the nucleolus targeting sequence, deletion mutations within the 20 amino acids of protein alpha were constructed. It was found that the deletion of (23)RRR(25), (29)RRR(31), or (23)RRRANNRRR(31) prevented the accumulation of EGFP fusion proteins into the nucleolus, demonstrating that (23)RRRANNRRR(31) contain the signal required for nucleolar localization. A similar distribution pattern of localization of protein alpha and its deletion mutants in SB and Cos-7 cells suggested that N-terminal residues of protein alpha (23)RRRANNRRR(31) constitute a nucleolus localization signal that functions in both fish and mammalian cells.     

Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.     

The lactate dehydrogenase-elevating virus capsid protein is a nuclear–cytoplasmic protein

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However, nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus, has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their normal cytoplasmic distribution. In the present study, the N protein of lactate dehydrogenase-elevating virus (LDV) of mice was examined for nuclear localization. The subcellular localization of LDV-N was determined by tagging N with enhanced green fluorescence protein (EGFP) at the N- and C-terminus. Both N-EGFP and EGFP-N fusion proteins localized to the nucleus and nucleolus of gene-transfected cells. Labeled N also accumulated in the perinuclear region, the site of virus replication. The LDV-N sequence contains a putative ‘pat4’-type nuclear localization signal (NLS) consisting of 38-KKKK. To determine its functional significance, a series of deletion constructs of N were generated and individually expressed in cells. The results showed that the ‘pat4’ NLS was essential for nuclear translocation. In addition, the LDV-N interacted with the importin-α and -β proteins, suggesting that the LDV-N nuclear localization may occur via the importin-mediated nuclear transport pathway. These results provide further evidence for the nuclear localization of N as a common feature within the arteriviruses.     

核定位信号类短肽的生物医学应用

核蛋白定位信号（nuclear localization signal,NLS）是一段富含精氨酸、赖氨酸等碱性氨基酸的短肽,是介导蛋白通过核孔复合体入核的必要信号序列。NLS存在于真核细胞核蛋白及病毒蛋白中,鱼精蛋白是应用较为广泛的天然NLS类短肽。针对NLS能够介导DNA、蛋白质、纳米粒等进入细胞核的特点,国内外学者对其在生物医学中的应用进行了广泛研究。     

Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein

The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7_38-98 from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence (₇₆IRTLEDLLM₈₄) changed the localization of 2xEGFP-E7_38-98 from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.     

Functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association

PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 "reversion resistant" mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG--SKKKS and PG--S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.     

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.     

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.     

Characterization of the nuclear localization signal in the DNA helicase responsible for Bloom syndrome

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, photosensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS. The BLM protein is homologous to DNA helicase and has two basic amino acid clusters in its C-terminal region. Previously, we reported that the distal arm of these basic amino acids clusters in the BLM protein functioned as the nuclear localization signal (NLS) of the protein. In this study, we generated plasmid constructs for expression of enhanced green fluorescent protein (EGFP) fused with various BLM protein variants having a mutation with deletions or substitutions in the basic amino acid and analyzed the subcellular localization of the expressed proteins. The EGFP-fused protein containing the basic amino acid cluster region proximal to the C-terminus of BLM helicase was localized exclusively in the nucleus. However, the EGFP-BLM proteins that lacked either Arg1344 or Lys1346 distributed in both the cytoplasm and the nucleus equally. Deletion of Arg1347 also resulted in localization in both the nucleus and cytoplasm, and substitution of Arg1344, Lys1346, Arg1347 or Arg1348 with non-basic amino acids reduced the nuclear localization of BLM protein. Mouse BLM protein which also migrate to the nucleus has two basic amino acid clusters in the C-terminus and the basic amino acids (Lys1346-Pro1347-Lys1348-Arg1349-Arg1350) proximal to the C-terminus are conserved between mouse and human. These findings suggest that the Arg1344-Ser1345-Lys1346-Arg1347 sequence at the C-terminus of the human BLM protein is essential for nuclear localization of this protein.     

Nuclear localization of the spinocerebellar ataxia type 7 protein, ataxin-7.

Spinocerebellar ataxia type 7 (SCA7) belongs to a group of neurological disorders caused by a CAG repeat expansion in the coding region of the associated gene. To gain insight into the pathogenesis of SCA7 and possible functions of ataxin-7, we examined the subcellular localization of ataxin-7 in transfected COS-1 cells using SCA7 cDNA clones with different CAG repeat tract lengths. In addition to a diffuse distribution throughout the nucleus, ataxin-7 associated with the nuclear matrix and the nucleolus. The location of the putative SCA7 nuclear localization sequence (NLS) was confirmed by fusing an ataxin-7 fragment with the normally cytoplasmic protein chicken muscle pyruvate kinase. Mutation of this NLS prevented protein from entering the nucleus. Thus, expanded ataxin-7 may carry out its pathogenic effects in the nucleus by altering a matrix-associated nuclear structure and/or by disrupting nucleolar function.     

A nuclear localization signal and a membrane association domain contribute to the cellular localization of the Tobacco mosaic virus 126-kDa replicase protein

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.     

Nucleolar localization of SmMAK16 protein from Schistosoma mansoni is regulated by three distinct signals that function independent of pH or phosphorylation status

SmMAK16 from the trematode Schistosoma mansoni is a protein that is known to localize in the nucleolus. Recent findings show that SmMAK16 is involved in 60S ribosomal subunit synthesis. Although the SmMAK16 protein contains putative nuclear localization signals (NLS), little is known about their precise function, redundancy or regulation. The goal of the current study was to identify and characterize the presence and functional regulation of the localization signals in SmMAK16. The SmMAK16 coding sequence and specific fragments were individually cloned in-frame into the pEGFP-C2 expression vector to encode Green Fluorescent Protein (GFP) fusion proteins. Constructs were individually transfected into COS-7 cells and fluorescent microscopy used to determine the cellular location and thus the presence of signals regulating nuclear and nucleolar localization. SmMAK16 was found to contain two NLSs and one nucleolar localization signal (NoLS). One of the signals contains a sequence identical to an established nucleolar detention signal that reportedly functions only under acidic cellular conditions. The localization of the SmMAK16-GFP constructs was analyzed under acidic conditions; however, altering pH did not influence the localization of SmMAK16. It has been previously reported that casein kinase 2 (CK2) can phosphorylate SmMAK16 at serines adjacent to one of the NLSs. One of these CK2 sites and the adjacent NLS are conserved with that of the SV40 Large T Antigen (LTA) and phosphorylation of this site in the SV40 LTA regulates the kinetics of the NLS. To discover if kinetic regulation also occurs in SmMAK16, mutant and wild type SmMAK16-GFP proteins were purified and injected into individual COS-7 cells. No difference in the rate of transport was found between wt and mutant SmMAK16 proteins. Therefore, SmMAK16 localizes to the nucleolus using three separate signals, two NLSs and one NoLS, however, these signals appear to function independently of pH and phosphorylation by CK2.     

Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.

Human papillomavirus type 16(HPV16) L1 and L2 capsid proteins can be detected only in the nucleus of infected cells. For other nuclear proteins, specific sequences of basic amino acids(aa) termed nuclear localization signals (NLS) direct the protein from the cytoplasm to the nucleus. We used a series of deletion and substitution mutations of the HPV16 L1 protein, produced by recombinant vaccinia virus (rVV), to identify NLS within HPV16 L1 and showed that HPV16 L1 contains two NLS sequences, each containing basic aa clusters. One NLS consisted of 6 basic amino acids (KRKKRK from aa 525 to 530) at the carboxy terminal end of L1. The other NLS contained 2 basic aa clusters(KRK from aa 510 to 512 and KR at aa 525, 526) separated by 12 amino acids. Mutations in either NLS did not alter nuclear localization of L1 when the other remained intact, but mutations to both prevented nuclear localization of L1. The L1 NLS could be overridden by introduction of a membrane binding sequence at the amino terminal end of the protein. A databases search showed that all sequenced papillomaviruses are predicted to have L1 and L2 capsid proteins with sequences of basic amino acids homologous with one or both NLS of HPV16 L1.     

Intracellular localization of the UL31 protein of herpes simplex virus type 2

Summary.  We studied intracellular localization of the UL31 protein of herpes simplex virus type 2 (HSV-2) in infected and transfected cells. The UL31 protein localized diffusely throughout the nucleus in infected Vero cells and the distribution patterns of the UL31 protein appeared to be different from those of either replication protein ICP8 or capsid protein ICP35. In transfected Vero cells it localized diffusely throughout the nucleus except the nucleolus at early times after transfection. At very low efficiency, it accumulated in the nucleolus. At intermediate times after transfection, the UL31 protein showed punctate staining in the nucleus. These punctate forms fused and became larger. At later times after transfection, granular forms further fused and a nuclear diffuse pattern virtually disappeared. We also constructed five N and C terminal deletion mutants of the UL31 protein for transfection assays and showed that the region containing amino acids 44 to 110 was important for nuclear and nucleolar localization. Moreover, green fluorescent protein (GFP)-targeting experiments showed that the UL31 protein was able to transport nonnuclear GFP to the nucleus and nucleolus as a fusion protein. Accepted May 10, 1999 Received April 16, 1999     

Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNAβ satellite

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of geminiviruses. Capsid proteins (CPs) of geminiviruses are multifunctional proteins thought to be involved in this process. The CP of monopartite geminiviruses is absolutely essential for virus movement. To more precisely examine the role of CP, we have constructed a series of single and double deletions into the coding sequence of Tomato leaf curl Java virus (ToLCJAV) CP and examined sub-cellular localization using transient expression of GFP fusion proteins. In this report, the domains of the CP encoded by ToLCJAV localized in the nucleus/nucleolus and cytoplasm in transfected cells were mapped. Deletion analysis revealed that the Arg-rich cluster from amino acids (aa) ¹⁶KVRRR²⁰ in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). The region from aa ⁵²RKPR⁵⁵ contained basic amino acid cluster was capable to redirect the CP to the nucleus. Further, both transient expression and yeast hybrid assays demonstrated that CP was capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa (²⁴⁵LKIRIY²⁵⁰) reside at C-terminal part of CP. This hydrophobic region caused transport of GFP to the cytoplasm. However, ToLCJAV CP NLSs and NES show peculiarities in the number and position of basic residues. Taken together, these results demonstrated that ToLCJAV CP shuttles between the nucleus and cytoplasm, such an activity homolog to bipartite geminivirus BV1 ORF.     

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.