Infection by agnoprotein-negative mutants of polyomavirus JC and SV40 results in the release of virions that are mostly deficient in DNA content
© Sariyer et al; licensee BioMed Central Ltd. 2011
Received: 19 January 2011
Accepted: 24 May 2011
Published: 24 May 2011
Human polyomavirus JC (JCV) is the etiologic agent of a brain disease, known as progressive multifocal leukoencephalopathy (PML). The JCV genome encodes a small multifunctional phospho-protein, agnoprotein, from the late coding region of the virus, whose regulatory functions in viral replication cycle remain elusive. In this work, the functional role of JCV and SV40 agnoproteins in virion release was investigated using a point mutant (Pt) of each virus, where the ATG codon of agnoprotein was mutated to abrogate its expression.
Analysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content.
The results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.
List of abbreviations
Simian vacuolating virus 40
Progressive multifocal encephalopathy
Phosphate buffered saline
Dulbecco's modified eagle's medium
large T antigen, Pt: point mutant
Hanks balanced salt solution
Primary human fetal glial cells
Fetal bovine serum.
A large number of studies indicate that the small regulatory proteins of many viruses play important roles in various aspects of viral infection cycle, including replication [1–3], transcription [4–10], translation , export of viral transcripts from nucleus to cytoplasm , viral assembly  and release of viral particles [14, 15]. In addition, these proteins may also modulate host-cell functions by deregulating the expression of key cellular genes . Therefore, such regulatory proteins are important for successful completion of the viral life cycle and study of their regulatory roles in viral life cycle is critically important for understanding of the viral replication process and the disease progression that respective viruses induce in their host.
The late coding region of human polyomavirus JC (JCV) and simian virus 40 (SV40) encodes a small regulatory phosphoprotein, agnoprotein, whose expression during the viral lytic cycle has been demonstrated by biochemical and immunocytochemical methods [17–19]. Agnoprotein is a cytoplasmic protein predominantly localized to the perinuclear region of infected cells. A small amount of agnoprotein is also detected in nucleus in the infected cells. The expression pattern of agnoprotein in tissue sections from progressive multifocal leukoencephalopathy (PML) has also been analyzed and also shown to localize to the cytoplasmic and perinuclear regions of the infected brain cells from PML patients . Amino acid sequence alignment of the agnoproteins for JCV, BKV and SV40 shows a high degree of sequence identity of about 70% [10, 21]. While the amino-terminal and central regions of each agnoprotein exhibit considerable sequence identity with one another, sequences toward the carboxy-terminal region are more divergent.
JCV is the etiologic agent of the fatal demyelinating disease of the brain, PML [7, 22–25] and its late gene product, agnoprotein, has been previously shown to functionally interact with other JCV regulatory proteins, including large T-antigen  and small t-antigen  and several cellular factors [16, 19]. In addition, agnoprotein has been shown to have inhibitory effects on cell cycle progression . Mutational analysis of agnoprotein from the closely related virus SV40 suggested that it may have effects on various aspects of the viral lytic cycle including transcription, translation, virion production and maturation of the viral particles [27–34]. It has been known for more than a decade that SV40 and BKV agnoproteins are phosphorylated but no function has yet been assigned to this modification [18, 35]. More recent studies explored the possibility that potential phosphorylation sites of agnoprotein are the targets for well-characterized protein kinases, including protein kinase C (PKC). Indeed, these studies demonstrated that agnoprotein is phosphorylated by PKC and phosphorylation turns out to play a significant role in the function of this protein during the viral replication cycle [36, 37]. More recent reports also showed that agnoprotein deletion mutants are non-functional but can be rescued by trans-complementation [36, 38]. In addition, it has been suggested that agnoprotein aids in the release of virions from infected cells .
In order to delineate whether agnoprotein is involved in release of viral particles from infected cells, we have utilized point mutants of JCV and SV40 agnoproteins in which the ATG translation initiation codon of agnogene was altered and thereby the expression of the protein was ablated. In this report, we provide experimental evidence indicating that both JCV and SV40 virions are efficiently released from the infected cells in the absence of agnoprotein, however, the released viral particles are mostly deficient in DNA content, which greatly hampers the ability of the propagation of the mutant virus relative to wild-type.
Primary human fetal glial (PHFG) cells were prepared as follows: Brain tissue from an aborted 16-week-old fetus was first cut into small pieces in Hanks balanced salt solution (HBSS) and the clumps were mechanically disrupted by repeated pipetting of the soft tissue. To further separate the cells, the tissue was incubated at 37°C with trypsin (0.005%) and DNAse I (50 μg/ml) for 30 min. Fetal bovine serum (0.1%) was then added to inactivate trypsin and the cells were washed with HBSS twice to remove trypsin. The cells were then passed through a 70 mm mesh to remove larger cell clumps. The cells in the filtrate were spun down and the pellet was resuspended in a small volume of HBSS with a fire polished glass pipette to obtain single cells. After cell viability and count were determined, cells were resuspended in growth media D-MEM+F12 containing fetal bovine serum, FBS (10%), L-glutamine (2 mM), insulin (2.5 mg/l) and gentamycin (50 mg/l). The cells were plated on collagen-coated flasks at a concentration of 2-10 × 106 per 75 cm2 flask and incubated at 37°C. SVG-A is a subclonal population of a human glial cell line which was established by transformation of human fetal glial cell line with an origin-defective SV40 mutant . SVG-A cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin, 100 μg/ml). They were maintained at 37°C in a humidified atmosphere with 7% CO2. Cos-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (FBS) and antibiotics (penicillin/streptomycin, 100 μg/ml). They were maintained at 37°C in a humidified atmosphere with 7% CO2.
Stable cell lines
SVG-A cells (2 × 105 cells/100 mm tissue culture dish) were either stably transfected with pcDNA3-JCV agnoprotein expression plasmid (pcDNA3-JCV-Agno) (5 μg/plate)  or pcDNA3 vector alone (Invitrogen) (5 μg/plate) by lipofectin method according to manufacturer's recommendations (Invitrogen). At five hour posttransfection, transfectants were washed twice with phosphate buffered saline (PBS) and refed with DMEM containing 10% FBS. After twenty-four hours, cells from each 100-mm plate were trysinized and replated onto ten 100-mm plates. Cells were allowed to attach for six hours and then the medium was replaced with DMEM containing 400 μg/ml G418 and 10% FBS. The medium was changed every three days until individual colonies formed. Individual colonies were randomly selected, screened for agnoprotein expression by Western blotting, expanded and frozen in liquid nitrogen.
Description of both JCV WT (Bluescript KS+JCV-Mad-1 WT) and JCV agnoprotein point mutant (Bluescript KS+JCV-Mad-1 Pt) was previously described . Simian virus 40 genome [SV40(776)] was subcloned from pBR322 vector into Bluescript KS+ at the BamH I site and designated as Bluescript KS+SV40(776) WT. SV40 agnoprotein Pt mutant at ATG site was created by an "Excite™ PCR-based site-directed mutagenesis Kit" (Stratagene) utilizing the following primers: Primer 1. SV40(776) (331-362): 5'-GG AGA TCT TGC TGC GCC GGC TGT CAC GCC AGG-3'. Primer 2. SV40(776) (330-309): 5'-TGA AAT AAC CTC TGA AAG AGG-3'. Underlined sequence represents the substitution of Nco I site with a newly created Bgl II site. PCR was performed according to manufacturer's recommendations. Briefly, 10 ngs of Bluescript KS+SV40(776) WT plasmid were PCR amplified with 15 picomols of each primer shown above under stringent reaction conditions. Primers were phosphorylated by T4 polynucleotide kinase at 5'-ends before PCR reaction. After initial denaturation of the plasmid for 10 min at 94°C, reaction was cycled 12 times under the following parameters: 95°C, 2 min; 56°C, 2 min; and 72°C 8 min; and 72°C 10 min for one cycle. Upon PCR reaction, the PCR product was treated with Dpn I enzyme to eliminate template DNA, religated and transformed into supercompetent Epicurian Coli XL-1-Blue supercompetent cells (Stratagene). In the point mutant construct, Nco I site (CCATGG) of WT strain containing the translation initiation codon (ATG) of agnoprotein was converted into Bgl II site (AGATCT) by base substitution which allowed us to distinguish the mutant from WT by restriction enzyme digestion. Finally, base substitutions and overall integrity of the viral DNA sequences were verified by DNA sequencing for SV40 Pt and JCV Pt plasmids. pcDNA3-JCV-Agnoprotein expression plasmid (pcDNA3-JCV-Agno) was described previously .
Both PHFG and SVG-A cells (2 × 106 cell/75 cm2 flask) were transfected either with Mad-1 WT (8 μg) or Mad-1 Pt (8 μg) mutant JCV viral DNA using lipofectin-2000 according to manufacturer's recommendations (Invitrogen). CV-1 cells (1.5 × 106 cell/75 cm2 flask) were transfected either with SV40(776) WT (8 μg) or SV40(776) Pt (8 μg) mutant viral DNA using lipofectin-2000. At five hour posttransfection, cells were washed twice with PBS. Cos-7 cells were transfected as described for CV-1 cell lines. PHFG cells were fed with D-MEM+F12 containing FBS (10%), L-glutamine (2 mM), insulin (2.5 mg/l) and gentamycin (50 mg/l); and SVG-A, Cos-7 and CV-1 cells were fed with DMEM supplemented with 5% FBS.
Whole-cell extracts prepared from PHFG cells untransfected or transfected with either WT JCV Mad-1 genome or JCV Mad-1 Pt mutant genome at 7d, 14d, and 21d posttransfection were resolved on SDS-15% PAGE and blotted onto nitrocellulose membranes. Blotted membranes were probed with an anti-agnoprotein polyclonal antibody as described previously  and detected by a chemiluminescence (ECL) method according to the manufacturer's recommendations (Amersham). In parallel, nuclear extracts prepared from either transfected or untransfected cells were analyzed for viral VP1 and large T antigen (LT-Ag) by Western blotting utilizing a monoclonal anti-VP1 antibody (PAb 597) (a kind gift from Dr. W. Atwood, Brown University, Rhode Island) and a monoclonal anti-SV40 LT-Ag antibody (Ab-2), which is cross-reactive with JCV LT-Ag respectively. Similarly, whole-cell and nuclear extracts prepared from SVG-A cells stably expressing JCV agnoprotein which were either transfected with JCV Mad-1 WT or JCV Mad-1 Pt mutant were analyzed for agnoprotein. In addition, whole-cell and nuclear extracts were prepared from CV-1 cells untransfected or transfected with either SV40(776) WT genome or SV40(776) Pt mutant genome at indicated time points posttransfection. Extracts were then analyzed for SV40 agnoprotein, LT-Ag and VP1 expression by Western blotting using anti JCV agnoprotein, anti-SV40 LT-Ag (Ab-2) and anti-JCV VP1 (PAb597) antibodies. Western blots were probed with an anti-lamin A antibody to demonstrate the equal loading of the protein extracts.
Replication assays were carried out as previously described . Briefly, PHFG cells or SVG-A cells (2 × 106 cells) grown in 75 cm2 flasks were transfected either with JCV Mad-1 WT or JCV Mad-1 Pt mutant viral genome (8 μg/2 × 106 cells/75 cm2 flask) by lipofectin-2000 transfection method. Of note, The pBluescript (back bone) vector from both JCV and SV40 plasmids was digested with BamH I before using the plasmids in transfections. Lipofectin-DNA mixture was incubated with cells for 5 h and washed with PBS. Transfected SVG-A cells were fed with complete D-MEM media with 3% FBS. Transfected PHFG cells were fed with a special growth media D-MEM+F12 media containing 10% FBS, L-Glutamine (2 mM), gentamycin (50 mg/l) and insulin (2.5 mg/l). At indicated time points posttransfection, low-molecular-weight DNA containing both input and replicated viral DNA was isolated by the Hirt method , digested with BamH I and Dpn I enzymes, resolved on a 1% agarose gel and analyzed by Southern blotting.
Indirect immunofluorescence microscopy
Indirect immunofluorescence microscopy studies were performed as previously described [19, 42, 43]. Briefly, CV-1 cells transfected with either SV40(776) WT or its Pt mutant genome were seeded at subconfluency on polylysine-coated glass chamber slides at 5th day posttransfection. The next day, cells were washed twice with PBS and fixed in cold acetone. Fixed cells were blocked with 5% bovine serum albumin in PBS for 2 h and incubated with a monoclonal primary anti-SV40 large T antigen (Ab-2) antibody overnight. Cells were subsequently washed four times with PBS-0.01% Tween 20 for 10-min intervals and incubated with an anti-mouse fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody for 45 min. Finally, the slides were washed three times with PBS, mounted, and examined under fluorescence microscope for detection of LT-Ag. Indirect immunofluorescence microscopy studies were performed for JCV Mad-1 WT and its Pt mutant. SVG-A cells transfected with either JCV Mad-1 WT or its Pt mutant genome were seeded at subconfluency on polylysine-coated glass chamber slides at 6th day posttransfection and processed for detection of agnoprotein and VP1 as described above for SV40 using anti-Agno polyclonal  and anti-VP1 monoclonal (PAb597) primary antibodies followed by incubation with a FITC-conjugated goat anti-rabbit and Rhodamine-conjugated goat anti-mouse secondary antibodies respectively as described in each figure legend. Indirect immunofluorescence microscopy studies were also performed for SV40(776) WT and its Pt mutant in Cos-7 cells as described in the respective figure legend.
Viral particle release assay
Mad-1 WT genome or its agnoprotein Pt mutant were separately transfected into SVG-A cells (8 μg DNA/2 × 106 cells/75 cm2 flask). Supernatants from infected cells were collected at indicated time points, centrifuged at 16,000×g to clear cell debris and were subjected to immunoprecipitation using an anti-VP1 antibody (PAb597) (2 μg). Half of the samples were analyzed by Western blotting using anti-VP1 antibody (PAb597), the other half was analyzed by Southern blotting for detection of encapsidated viral DNA. The viral DNA from capsids was purified employing Qiagen spin columns , digested with BamH I and Dpn I enzymes, resolved on an 1% agarose gel and analyzed by Southern blotting using probes prepared from whole Mad-1 genome. In parallel, SV40(776) WT or its Pt mutant DNA was transfected into CV-1 cells (8 μg/2 × 106 cells/75 cm2 flask) and supernatants of the samples were collected at indicated time points and processed as described above for JCV Mad-1 WT and its Pt mutant.
Both viral early and late protein expression are markedly reduced in the absence of agnoprotein
In the case of SV40, we also assessed the effect of agnoprotein on viral early gene expression using its Pt mutant genome. CV-1 cells, which are permissive for SV40 replication, were transfected with either SV40(776) WT or its Pt mutant genome. Nuclear extracts were prepared at the time points indicated and analyzed by Western blotting for the expression of SV40 LT-Ag. As shown in Figure 1B, expression of LT-Ag from both WT and the mutant backgrounds 24h (1d) after transfection (upper panel, lane 2) was readily detectable but the level of SV40 LT-Ag expression in cells transfected with the Pt mutant genome decreased significantly (compare lane 3 with 2). A similar expression profile was observed for both mutant and WT viruses even at 3d after transfection. Interestingly, at 6d and 9d after transfection, whereas the level of LT-Ag expression from WT background gradually but substantially increased (lanes 6 and 8), that of LT-Ag from the Pt mutant background reduced to barely detectable levels (lanes 7 and 9). In parallel, we analyzed the presence and absence of agnoprotein expression in whole cell extracts from SV40 WT and SV40 Pt mutant backgrounds respectively (Figure 1B, bottom panel). While the expression of SV40 agnoprotein was readily detectable in CV-1 cells transfected with WT genome (lanes 4, 6 and 8), as expected, its expression was not evident in cells transfected with SV40 Pt mutant genome (lanes 3, 5, 7 and 9).
The effect of SV40(776) Pt mutant on viral late gene expression was also analyzed. Nuclear extracts prepared from CV-1 cells transfected with either SV40(776) WT or the Pt mutant genome were analyzed by Western blot for VP1. As shown in Figure 4B, the level of SV40 VP1 expression, although demonstrated a relatively low but steady expression for 1 and 3 days after transfection (lanes 2-5) followed by a very strong increase at 6 and 9 days for WT (lanes 6 and 8 respectively). In contrast, Pt mutant showed a declining course after 3 days where the level of VP1 expression dropped to undetectable levels at 6 days and 9 days (lanes 7 and 9 respectively) similar to the pattern of early gene expression described above (Figure 1B, upper panel). Collectively, all these results suggest that, in the absence of agnoprotein, JCV and SV40 protein expression (early and late) appear to be severely down-regulated.
Replication efficiency of JCV and SV40 greatly diminished in the absence of agnoprotein
We next examined the replication properties of the SV40 Pt mutant compared to WT in CV-1 monkey kidney cells to see if it behaves in a similar manner observed for JCV. The results from this replication studies showed that the replication efficiency of the SV40 Pt mutant was also hampered compared to the WT (Figure 5B). The level of replicated DNA from WT virus gradually increased during the course of the infection cycle until 9th day posttransfection. However, the amount of replicated DNA for SV40 Pt mutant virus declined after 3th days following transfection reaching a point where its replication was barely detectable after day 6 of the infection cycle, which is consistent with the results obtained from viral protein expression studies in Figure 1B. The quantitative analysis of the results showed that there was approximately 120-fold more replicated DNA for the WT compared to the mutant at day 9 posttransfection.
Stable expression of agnoprotein in trans restores the replication activity of the JCV Pt mutant virus
Analysis of the virions released from SVG-A cells transfected/infected with JCV Pt mutant
Analysis of the virions released from CV-1 cells transfected/infected with SV40 Pt mutant
Analysis of growth properties of SV40 Pt mutant in Cos-7 cells, constitutively expressing SV40 large T antigen
The regulatory roles of agnoprotein in JCV life cycle are not fully understood. In this work, we specifically investigated the regulatory role(s) of JCV and SV40 agnoprotein in virion release utilizing agnoprotein point mutants of each virus in which the ATG initiation codon has been removed so as to ablate expression. We found that both JCV and SV40 viral gene expression and replication were drastically reduced in the absence of agnoprotein suggesting that both viruses require agnoprotein for the successful completion of their lytic cycle. Our findings are also supported by a recent report by Myhre et al, in which it was demonstrated that a non-functional BKV mutant with deletions in the agnogene can be complemented in trans from a co-existing BKV rr-NCCR variant .
In addition, consistent with our findings from gene regulation studies, JCV and SV40 agnoprotein Pt mutants showed an enormous negative effect on viral DNA replication. SVG-A cells used in replication assays were derived from PHFG cells by stably transfecting a replication defective SV40 genome . This cell line expresses the viral early regulatory protein, LT-Ag, but not the viral late genes including agnoprotein. Preparation of PHFG cells is labor intensive, costly, and there is variability between the batches of cell preparations, greatly affecting consistency of the results. SVG-A cells constitutively express SV40 LT-Ag, which was previously shown to cross-regulate JCV replication [45–48], but our data presented in this work convincingly showed that expression of SV40 LT-Ag alone is not sufficient for the efficient replication of neither JCV Pt mutant in SVG-A cells (Figure 5A) nor SV40 Pt mutant in Cos-7 cells (Figure 9), which also constitutively express SV40 LT-Ag. It should be noted here that the origin of DNA replication of JCV and SV40 in agnoprotein Pt mutants was intact, which was verified by sequencing. As such, the relatively low level of replication of JCV and SV40 Pt mutants compared to their WTs cannot be attributed to the unintended mutations that occurred during the mutagenesis process, which greatly affect the replication of each mutant. Therefore, it was expected that SV40 LT-Ag would up-regulate JCV replication in agnoprotein Pt mutant background regardless of the absence of agnoprotein. However, we found that agnoprotein is required for efficient regulation of JCV and SV40 replication in the context of the whole viral genome, i.e., the constitutive expression of SV40 LT-Ag is not sufficient to complement the absence of agnoprotein for the efficient replication of JCV and SV40. In fact, the results from the complementation assays support this conclusion and demonstrate that the level of replication of the JCV agnoprotein Pt mutant is alleviated to a level comparable to that of WT when agnoprotein is provided to the replication system in trans (Figure 6A).
We also investigated the question of whether agnoprotein has a role in the release of viral particles and found that JCV and SV40 virions are efficiently released from the infected cell in the absence agnoprotein, but they are mostly deficient in viral DNA content (Figure 7B and 8B). Our findings are in contrast with the recent report by Suzuki et al.  which suggests that agnoprotein functions as a viroporin and therefore may play a role in release of virions from JCV-infected cells. Our results do not support this idea since we found that viral particles were efficiently released from infected cells to the culture medium in the absence of agnoprotein. However, the released virions are not as infectious particles as those of WT due to the lack of viral encapsidated DNA, i.e., they appear to be mostly composed of empty capsids and therefore unable to initiate an efficient next round of the infection cycle. This may be the major reason why the level of propagation of agnoprotein Pt mutant is profoundly reduced compared to that of WT.
The small regulatory proteins of many viruses play important roles in regulation of the viral life cycle and are therefore critical to the fine tuning of many aspects of virus-host interactions. Our findings in this report indicate that the agnoprotein of JCV and SV40 is required for efficient regulation of viral DNA replication and gene regulation. This may have functional consequences for the successful completion of the JCV lytic cycle. In light of our findings, we propose that agnoprotein may substantially contribute to the process of proportional production of viral capsid proteins versus that of viral DNA, thereby leading to the formation of a high number of optimally infectious viral particles for the next round of the infection cycle. Finally, given the fact that JCV is the etiologic agent of PML and may be involved in the induction of some of the human malignancies, such findings make agnoprotein an attractive target for therapeutic approaches to control JCV-induced diseases in the affected individuals. Understanding the molecular mechanisms associated with the regulatory functions of agnoprotein is important for unraveling the molecular secrets of the unique biology of JCV and JCV-associated diseases.
We would like to thank past and present members of the Department of Neuroscience and Center for Neurovirology for their insightful discussion and sharing of ideas and reagents. This work was made possible by grants awarded by NIH to MS.
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