Packaging of viral RNAs in virions of adenoviruses
© Xing and Tikoo; licensee BioMed Central Ltd. 2009
Received: 22 December 2008
Accepted: 05 February 2009
Published: 05 February 2009
Earlier, we detected viral RNAs packaged in the porcine adenovirus (PAdV) -3 virions. Using Southern blot analysis, we further demonstrated that the viral RNAs were predominantly packaged in CsCl purified mature capsids (containing viral genome) than empty/intermediate capsids. Some of the packaged viral RNAs appear to be polyadenylated. Real-time reverse transcription (RT)-PCR analysis indicated that the copy number of the tested viral mRNAs encoding E1Bsmall and fiber proteins was less than one per full capsid. Moreover, detection of viral RNA packaged in CsCl purified human adenovirus (HAdV) -5 virions indicates that the viral RNA packaging might be a common phenomenon in members of Adenoviridae family. Further quantitative analysis of viral protein, DNA, and RNA in CsCl purified mature and empty/intermediate capsids of recombinant HAdV-5 expressing enhanced green fluorescent protein indicated that the traceable viral RNA detected in empty/intermediate capsids seems associated with the presence of traceable viral genomic DNA. Taken together, our data suggest that the viral RNAs may be passively packaged in adenovirus virion during encapsidation of viral genomic DNA in cell nuclei. Thus, viral RNA packaging may be a characteristic feature of adenoviral genomic DNA encapsidation.
The phenomenon of encapsidation of viral RNAs was initially uncovered in members of Herpesviridae family [1–4]. These RNAs could be translated into proteins that would function prior to de novo transcription from the viral genome [1, 2, 5]. Alternatively, these viral RNAs might facilitate organizing the structure of the tegument domain through RNA-protein interactions during virion assembly  as found in retroviruses [7, 8]. Moreover, the studies with human cytomegalovirus (HCMV) suggested that both the viral and the cellular RNAs were nonspecifically incorporated into the virions through interactions with some virion proteins during budding . However, studies with herpes simplex virus (HSV) -1 and Kaposi's sarcoma-associated herpesvirus (KSHV) suggested that some virion RNAs were specifically incorporated into the virions [1, 4].
Adenoviruses are another family of DNA viruses that infect a wide variety of mammals and birds . Adenovirus is non-enveloped containing a single, linear double-stranded DNA of approximately 26–43 kb within an icosahedral capsid of 70–100 nanometer in diameter . The assembly of mature adenovirus virion leads to the formation of intermediate capsids, some of which may contain little viral or cellular DNA [11–13]. Earlier, we demonstrated that viral RNAs were packaged in porcine adenovirus (PAdV) -3 virions, a non-enveloped DNA virus . Another report suggested that a non-viral RNA (LacZ mRNA) transcribed from a recombinant human adenovirus (HAdV) -5 is packaged into the HAdV-5 virions . In this report, we examined the incorporation of viral RNAs in mature and empty/intermediate capsids of PAdV-3 and HAdV-5. Moreover, the characteristics of packaged viral RNAs were further examined by Southern blot hybridization and real-time RT-PCR analysis.
Cells and Viruses
VIDO R1 cells  were grown in Eagle's minimum essential medium supplemented with 10% heat inactivated fetal bovine serum (FBS). The wild-type (wt) PAdV-3 (6618 strain)  and mutant Pav3-PL1  were propagated and titrated in VIDO R1 cells. 293 cells  were grown in Dulbecco's modified Eagle's medium supplemented with 5% FBS. The wild-type (wt) HAdV-5 and recombinant HAV5.EGFP containing enhanced green fluorescent protein (EGFP) gene inserted in E1 region of HAdV-5 were propagated and titrated in 293 cells.
Isolation of viral capsids
In order to obtain empty/intermediate and mature capsids, VIDO R1 cells were infected with wt PAdV-3 or mutant Pav3-PL1 at a multiplicity of infection (MOI) of 10 plaque forming units (PFUs). At 48 h post infection, the infected cells were collected and freezed-thawed three times. The cell lysates were subjected to a step gradient with 1.2 and 1.4 g of CsCl/ml and ultracentrifuged at 35,000 rpm for 2 h. Two major bands were harvested and loaded on a continuous CsCl gradient at 1.32 g/ml and centrifuged at 35,000 rpm overnight. The bands from this gradient were further purified on a third gradient and then dialyzed into phosphate-buffered saline (PBS). Similarly, HAdV-5 capsids were purified from 293 cells infected with wt HAdV-5 or recombinant HAV5.EGFP. Total RNA was isolated from purified capsids, or virus infected cells as described earlier .
CsCl-purified virions were adsorbed to nickel grids. After adsorption, the grids were stained with 1% solution of phosphotungstic acid for 60 s and examined by using transmission electron microscope (Philips EM410). Photographs were taken from representative areas from each sample.
The wt PAdV-3 genomic DNA was isolated from purified viral capsids as described earlier [14, 18]. Viral genomic DNA or pFHAV5 plasmid DNA containing E1A-deleted HAdV-5 genome was digested with Hind III and loaded into 1% agarose gel. Fractionated DNAs were transferred into Gene Screen Plus hybridization transfer membranes (PerkinElmer) and probed with [32P]-labeled cDNA probes prepared by reverse transcribing 2 μg of RNAs isolated from purified capsids or virus infected cells in the presence of [32P]-labeled dCTP as described earlier .
Real-time PCR was performed with Platinum Quantitative PCR SuperMix-UDG kit (Invitrogen) as instructed by manufacturer. The cDNAs were prepared by reverse transcription as described previously . The synthesized cDNAs or viral genomic DNAs were used as templates in real-time PCR, using iCycler real-time PCR system (Bio-Rad). TaqMan probes for E1Bsmall (5'-TTTATC AAGGTAGTAGCAG AGGCCA-3') and fiber (5'-TCCCTGGGTCCCGGTCT TTCTA ACT-3') were labeled with FAM at 5'-end and TAMRA at 3'-end, and purchased from Qiagen. The primers RTE1B1 (5'-AG TACA GGGGT CTCAGAACT-3') and RTE1B2 (5'-CTCCACAA A A ATCTCAATCA-3') are specific for PAdV-3 E1Bsmall, TRFIB1 (5'-GATGGCAAGCTGG TTCTC A A-3') and TRFIB2 (5'-GGAGCTGTGACTTGCAGA CT-3') are specific for PAdV-3 fiber . After incubation at 50°C for 2 min and 95°C for 2 min, the reaction was run for 45 cycles with denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s. As a negative control, RNase-treated RNA samples were also reversely transcribed and simultaneously subjected to PCR reaction. Serial 10-fold dilutions of known amount of plasmid pFPAV3 DNA (known as pPAV200) containing E1Bsmall and fiber genes were used as positive control to generate the standard curve. The copy numbers were calculated by converting the weight unit of double-stranded pFPAV3 DNA into the numbers of single-stranded DNA molecules. The averaged cycle threshold (Ct) values of five replicates were used to determine the relative RNA copy number.
Results and discussion
Preparation and characterization of PAdV-3 capsids
To further characterize the integrity of empty/intermediate and mature capsids, DNA was isolated from both bands. Ethidium bromide-stained agarose gel showed that the viral genomic DNA is present predominantly in the lower bands (data not shown), confirming that the viral particles with the density of 1.32 g/ml are the mature capsids. To determine the integrity of the capsids, transmission electron microscopy was performed as described . Analysis of the preparations by the electron microscope (EM) suggested that the mature and the empty/intermediate capsids of both wt PAdV-3 and mutant Pav3-PL1 are predominantly intact (Fig 1B, wild-type PAdV-3).
Viral RNA was mainly detected in mature capsids and appeared polyadenylated
To further characterize the viral RNAs packaged in PAdV-3 virions, reverse transcription (RT) of the RNAs from the mature capsids was primed with only oligo-dT of 12–16 nucleotides (nt), which is specific for mRNAs containing polyA tail. As a positive control, 2 μg of total RNA isolated from wt PAdV-3-infected VIDO R1 cells at late times post infection was reversely transcribed in the presence of [32P]-dCTP as described above. As a negative control, 2 μg of total RNA from swine testis (ST) cells was reversely transcribed in the presence of [32P]-dCTP. As expected, radiolabeled probes made from the RNAs isolated from uninfected ST cells did not produce any positive signals in Southern blot (Fig. 2, panel c). In contrast, radiolabeled probes made from RNAs isolated from wt PAdV-3-infected VIDO R1 cells resulted in strong signals, indicating that PAdV-3 specific RNAs were present in cellular RNA preparations (Fig. 2, panel b). No signal could be detected with radiolabeled probes made from the RNase treated RNA isolated from wt PAdV-3-infected VIDO R1 cells (Fig. 2, panel b). Compared with the oligo-dT and hexamer-doubly primed RT reaction (Fig. 2, panel a), the oligo-dT primed RT reaction displayed weaker positive signals (Fig. 2, panel b). Compared to the results obtained with total RNAs from the virus infected VIDO R1 cells, the oligo-dT primed RT of virion RNAs from purified mature capsids also produced weaker positive signals (Fig. 1B. panel f) than RT primed with both oligo-dT and hexamer (Fig. 2. panel d). These results suggested that some viral RNAs packaged in the virions appear to be polyadenylated.
Viral RNA was packaged at a low copy number into PAdV-3 mature capsids
To quantify the RNAs packaged in the virions, we performed real-time RT-PCR with Platinum Quantitative PCR SuperMix-UDG kit (Invitrogen). We choose to quantitate RNAs specific for an early region gene (E1Bsmall) and a late region gene (fiber) of PAdV-3. The viral genomic DNA and RNA were isolated from the same pool of purified mature capsids. Viral DNA was used as a template in real-time PCR to determine the total number of viral particles containing viral genome. The isolated RNAs with or without RNase treatment were reversely transcribed with the oligo-dT and hexamer as described above before using as templates in the real-time PCR to determine the RNA copy number based on the cycle threshold (Ct) values. Real-time PCR was performed by using Cycler real-time PCR system (Bio-Rad) as described in Methods.
Human adenovirus type 5 particles also contain viral RNAs
Viral RNA packaging appears to be associated with adenovirus genomic DNA encapsidation
Since both mature and empty/intermediate capsids may contain viral/cellular DNA [11–13], initially we quantitated the viral DNA in these capsids. Equal amounts (based on protein concentrations) of CsCl purified capsids were treated with RNase A and DNaseI as described above. The samples were further treated with 100 μg/ml proteinase K (Invitrogen) at 50°C for 2 h, before extracting sequentially with phenol and chloroform. The DNA was precipitated by isopropanol and dissolved in RNase A containing TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH8.0). Quantitative analysis suggested that mature capsids yielded 100 times more DNA than empty/intermediate capsids irrespective of the RNase/DNase treatment (Fig. 5B). The significant difference in the amount of DNA obtained from mature and empty/intermediate capsids is consistent with the general suggestion that mature capsid contains mature viral genomic DNA and empty/intermediate capsid may contain little premature viral DNA [11–13]. Moreover, DNA in mature and empty/intermediate capsids is not accessible to DNase/RNase treatment of the intact capsids. To confirm this further, we examined the expression of EGFP in 293 cells infected with equal amounts of mature or empty/intermediate capsid samples with or without RNase/DNase treatment. As seen in Fig. 5C, EGFP expression was detected in cells infected with mature or empty/intermediate capsids. However, empty/intermediate capsids transduced 104 fold less cells than the mature capsids. These results suggested that empty/intermediate capsids may contain premature viral and/or cellular DNA. Earlier reports have suggested that some of the intermediate capsids may contain left end of the adenovirus genome [11, 12].
To determine the identity of the RNAs isolated from RNase/DNase treated mature and empty/intermediate capsids, cDNA probes were synthesized using 2 μg of RNA isolated from mature or empty/intermediate capsids with RNase/DNase treatment and hybridized to Hind III-digested plasmid pFHAV5 containing E1A-deleted HAdV-5 genome. As seen in (Fig. 6B), these cDNA probes also resulted in positive signals, confirming that there are viral RNAs present inside the capsids. Although the total RNA yields from mature and empty/intermediate cpasids with RNase/DNase treatment appeared similar (Fig. 6A), the quantitative analysis indicated that the mature capsids contain more (30 fold) viral RNAs than empty/intermediate capsids (Fig. 6C). It is possible that the empty/intermediate capsids contain more cellular RNA. However, absence of detection of cellular mRNAs of house-keeping genes such as actin in virion RNAs  raises the possibility that the majority of the cellular RNAs isolated from mature and empty capsids might be rRNAs.
Although the virion RNAs were detected in the mature capsids, the low copy number of examined RNAs suggests that the viral RNAs might be packaged in a passive way by accompanying the viral genomic DNA at the stage of DNA encapsidation. So far, two models for adenovirus assembly have been proposed on the basis of pulse-chase analysis and the study of mutant viruses. The traditional model is that the adenovirus assembly begins with the formation of empty capsids, followed by the insertion of viral DNA into preformed empty capsids [24–26]. However, recently proposed model suggests that the capsids are assembled around the viral genomic DNA [27, 28]. The insertion of viral genome into the preformed empty capsids more likely would separate the synthesized RNAs from viral genome, resulting in missing of the viral RNAs in full capsids containing the viral genome. Thus, a mechanism of adenovirus assembly where packaging of virion RNAs with genomic DNA is coordinated with capsid formation appears to be plausible.
This work was supported by the grants from Natural Sciences and Engineering Research Council (NSERC) of Canada to SKT Published as VIDO article # 455.
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