Arch Virol (2012) 157:2227–2233 DOI 10.1007/s00705-012-1401-0

BRIEF REPORT

Development of a minigenome system for Andes virus, a New World hantavirus Kyle S. Brown • Hideki Ebihara • Heinz Feldmann

Received: 27 April 2012 / Accepted: 22 May 2012 / Published online: 21 July 2012 Ó Springer-Verlag 2012

Abstract The development of reverse genetics systems for negative-stranded RNA viruses is a rapidly evolving field that has greatly advanced the study of the many different aspects of the viral life cycle. Andes virus (ANDV) is a highly pathogenic hantavirus found in South America that causes hantavirus pulmonary syndrome but to date remains poorly characterized due to the lack of a reverse genetics system for genetic manipulation. Here, we describe the first successful minigenome system for a New World hantavirus, as well as many of the obstacles that still exist in the development of such a system.

The development of reverse genetic systems allows for changes to be made to a virus RNA genome using more easily manipulated and stable complimentary DNA (cDNA). Although the development of a full-length infectious clone system can be seen as the most desirable end goal, minigenome systems are typically established as a first step and have a number of advantages. Simply, the minigenome system allows for the analysis of the roles of

K. S. Brown
H. Feldmann Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada K. S. Brown
H. Feldmann National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada H. Ebihara
H. Feldmann (&) Division of Intramural Research, Laboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA e-mail: [email protected]

cis-acting sequences and trans-acting proteins on the noncoding regions (NCRs) of the virus genomes that are typically important in virus transcription, replication and genome packaging [5]. In general, the coding regions of the genome are removed and replaced with that of a single detectable reporter gene. This gene produces a product that can be assayed and quantified, producing an easy way to analyze how differences made in the NCRs affect expression without potential confounding factors that may be present in the coding regions of the genome [10]. In the case of high-containment pathogens, minigenome systems allow for handling in biosafety level 2. ANDV is a highly pathogenic, tri-segmented negativesense RNA virus from the genus Hantavirus, family Bunyaviridae [22, 26]. It causes hantavirus pulmonary syndrome (HPS) in South America, and human infection can result in a case fatality rate of up to 50 % [13, 14, 18]. The lack of sufficient molecular tools has severely stunted the investigation into the mechanisms of hantavirus pathogenicity. Although there have been minor successes in the development of some reverse genetics systems for Old World hantaviruses (found in Europe and Asia) [6, 24, 30] as well as other representatives of the family Bunyaviridae [1, 2, 7, 8, 17], similar attempts with New World hantaviruses have not been reported. Here, we show that we were able to successfully establish mammalian expression vectors for all of the ANDV proteins (nucleocapsid protein [N], glycoprotein precursor [GPC], co-translationally cleaved into GN and GC, and the RdRp protein [L]) using different promoters, in addition to ANDV minigenome vectors under the control of the human RNA polymerase I (Pol I) [31] or T7 bacteriophage RNA polymerase promoters [2, 4]. We also illustrate some of the difficulties that are still faced in the expanded development of hantavirus minigenome systems.

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The cDNAs containing the ANDV N and L open reading frames (ORFs) were derived from the Chilean isolate 9717869 [19], amplified using a RobusT One-Step RTPCR Kit (Finnzymes) and inserted into the subcloning vector pATX-MCS3 (a pBR322 derivative vector). All ANDV ORFs were amplified by PCR using a PfuTurbo PCR kit (Stratagene) with primers containing the appropriate nucleotide restriction sites for insertion into four different mammalian expression vectors: pCAGGS, containing a chicken b-actin (CAG) promoter [23]; pTM1, containing a T7 bacteriophage RNA promoter [20]; gWiz, containing a cytomegalovirus (CMV) immediate-early promoter (Genlantis); and pKS336, containing human elongation factor-1a (HEF-1a) promoter [25]. We also generated L protein expression plasmids containing a FLAG-tag (peptide sequence N-DYKDDDDK-C) at the Nor C-terminus of the ORF in order to detect the L protein due to the lack of available antibodies against this protein. The ANDV GPC ORF was also amplified by RT-PCR and inserted solely into the pCAGGS expression plasmid (Table 1). To construct the minigenome plasmids, the cDNAs containing the ANDV M-segment NCRs as well as

a small portion of the coding region (due to small size of the NCRs) were amplified by RT-PCR and inserted into pATX-MCS3. PCR amplification of the resulting vector was completed with primers to remove the coding-region sections and to add the chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP) or luciferase (LUC) reporter genes. The entire minigenome cassette containing a reporter gene flanked by the M-segment NCRs was cloned in the negative orientation (viral RNA [vRNA] sense) into two different expression vectors: one driven by a T7 bacteriophage RNA polymerase promoter (pT7-MCAT), and the other by a human RNA polymerase I (Pol I) promoter (pRF240-M-CAT) [6] (Table 1). For the detection of ANDV proteins, western blot analysis of cell lysates collected 48 hours post-transfection from Vero E6 or 293T cells transfected with ANDV recombinant protein expression plasmids using Fugene6 lipid transfection reagent (Roche) was carried out (Figure 1A). Samples were run on a 10 % SDS-PAGE gel and wet-transferred to Hybond-P PVDF membrane (GE Healthcare) as per standard protocols. Primary antibodies were diluted to 1:8,000 (mouse anti-GN, clone 6B9/F5,

Table 1 ANDV protein and minigenome expression plasmids Plasmids

Promoter

Expression WB

Expression IFA

MG Rescue ?

pCAGGS-N

CAG

???

???

gWIZ-N

CMV

???

???

??

pT7-N

T7

??

??

??

pKS336-N pCAGGS-NSslKO

HEF-la CAG

?? ???

?? ???

? ?

pCAGGS-L

CAG

-

-

?

gWIZ-L

CMV

-

-

??

pT7-L

T7

-

-

??

pKS336-L

HEF-la

-

-

?

pKS336-N-FLAG-L

HEF-la

-

?

-

pKS336-C-FLAG-L

HEF-la

-

-

-

pCAGGS-GPC (GN/GC)

CAG

???/???

??/??

?/?

pT7-M-CAT

T7

-----

-----

?

pRF240-M-CAT

Pol I

-----

-----

?

pT7-M-GFP

T7

-----

-----

-

pRF240-M-GFP

Pol I

-----

-----

-

pT7-M-LUC

T7

-----

-----

-

pRF240-M-LUC

Pol I

-----

-----

-

List of all plasmids used in the study, indicating the successful detection of protein by western blot (WB), immunofluorescent assay (IFA), or their use in minigenome rescue (MG). ANDV L expression constructs contained an N-terminal FLAG tag for detection by WB and IFA. ? or indicates successful/unsuccessful expression or use in minigenome rescue. - - - - - indicates no protein to be expressed. Key: CAG = chicken bactin promoter; CMV = cytomegalovirus immediate-early promoter; T7 = T7 bacteriophage RNA promoter; HEF-1a = human elongation factor-1a promoter; Pol I = human RNA polymerase I promoter; N = ANDV nucleocapsid protein; L = ANDV RNA-dependent RNA polymerase, GPC = ANDV glycoprotein precursor; NSs1KO = ANDV S-segment non-structural protein knockout; M-CAT = minigenome expression plasmid containing the M-segment non-coding regions (NCRs) and the chloramphenicol acetyl transferase (CAT) reporter gene. M-GFP = minigenome expression plasmid containing the M-segment NCRs and the green fluorescent protein (GFP) reporter gene; M-LUC = minigenome expression plasmid containing the M-segment NCRs and the luciferase (LUC) reporter gene

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Andes virus minigenome

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Austral Biologicals), 1:6,000 (mouse anti-GC, clone 2H4/ F6, Austral Biologicals) or 1:10,000 (mouse anti-N, clone 1A8/F6, Austral Biologicals). The secondary antibody (anti-mouse-HRP, Invitrogen) was diluted to 1:10,000. The secondary antibody was detected and imaged using an Amersham ECL Plus detection kit and ECL Hyperfilm (GE Healthcare). For IFA, cells were transfected as above and then fixed 48 hours post-transfection with an acetone:methanol mixture (1:1, v/v) (Figure 1B). Primary antibodies were diluted to 1:200 (mouse anti-GN), 1:200 (mouse anti-GC), 1:500 (mouse anti-N) or 1:1000 (mouse anti-FLAG, clone M2, Sigma-Aldrich). The secondary antibody (anti-mouse Alexa Fluor 488, Invitrogen) was diluted to 1:1,000. Cells were viewed and imaged using a fluorescent microscope (Zeiss Axiovert 200M) to confirm expression.

A

ANDV GPC

ANDV N 98

N-FLAG-ANDV L

98

64

kDa 50

Our results showed successful expression of the N protein in Vero E6 cells by both western blot (Figure 1A, left panel) and IFA (Figure 1B, left panel). Expression levels from the different promoter plasmids were similar, with only a slight decrease in expression from pTM1 and pKS336 (data not shown). We were also able to detect both glycoproteins in western blot (Figure 1A, middle panel) and IFA (Figure 1B, middle panel). We were unable to detect either of the FLAG-tagged L proteins through western blot using an anti-FLAG-antibody (Figure 1A, right panel), or by non immune staining methods such as Coomassie blue or silver staining. Detection of the N-terminal FLAG-tagged L was finally successful in IFA using the pKS336 expression plasmid, although at extremely low levels (Figure 1B, right panel). It is unknown why expression of the ANDV L protein was so inefficient, but

N

kDa 64 50

GN GC

No Detection

36 36

B

Fig. 1 ANDV protein expression. (A) Detection of ANDV protein expression by western blot (WB). L protein expression could not be detected. (B) Detection of ANDV proteins by immunofluorescent assay (IFA). Top panel – detection of ANDV N protein (left), glycoproteins GN & GC (middle) and L protein (right). Bottom panel – expression from corresponding negative control plasmids (empty

vector control). VeroE6 cells were used for the expression of the N protein and the glycoproteins; 293T cells were used for the expression of the L protein. Key: N = ANDV nucleocapsid protein; GN = ANDV N-terminal glycoprotein; GC = ANDV C-terminal glycoprotein; L = ANDV RNA-dependent RNA polymerase; kDa = kilodaltons

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only a few publications have yet described successful expression of a hantavirus L protein. The detection of the L protein in those cases has been achieved either indirectly through functional minigenome assays or directly as an L-GFP fusion protein by fluorescent microscopy or by antibodies produced from an L–specific peptide [6, 15, 30]. Despite including an N-terminal FLAG fusion tag on the L construct, we did not detect a protein in the expected 250-kDa range in western blot [15]. Detection was only possible in IFA, but because the tag is located at the N-terminus of the protein, there is no definite proof for fulllength L protein expression. Expression using a C-terminal FLAG fusion tag on the L protein was unsuccessful. A lack of expression or difficulties in detection of expression is likely due to a low rate of expression of this large protein, but issues with spliced or otherwise modified transcripts or fast proteasomal degradation of this large protein cannot be excluded. Furthermore, it is possible that the appropriate concentrations of other viral proteins may be necessary to help stabilize L protein expression, as previously shown with vesicular stomatitis virus L and P protein expression [3]. For rescue of ANDV minigenomes, the minigenome plasmids along with L and N (occasionally also GPC) expression plasmids were transfected into 6-well cell culture plates containing COS-7 cells using lipid transfection reagent transIT-LT1 (Mirus). Negative controls were carried out in the absence of ANDV protein expression plasmids or minigenome plasmids. The positive control consisted of the reporter gene expressed from a pCAGGS mammalian expression vector. T7 RNA polymerase expressed from a pCAGGS vector was included when using expression plasmids containing the T7 RNA promoter. The total amount of transfected DNA was normalized to the same value using an ‘‘empty’’ expression vector. Transfected cells were incubated 72 hours (optimized time point for expression) before analysis of the minigenome reporter signal. Attempts were made to view GFP and LUC minigenome reporter expression using fluorescent microscopy and luminescent assays, respectively, but were unsuccessful. In contrast, CAT minigenome reporter expression was detected from all plasmids. For the CAT assays, transfected cells were collected in PBS and briefly centrifuged to pellet cells. Cell pellets were subsequently lysed with Reporter Lysis Buffer (Promega). For the CAT reporter gene assay, samples were spun in a centrifuge to remove cellular debris, and 55 ll of the cell extract was then mixed together with 15 ll of FAST CAT substrate reagent (Molecular Probes) and 10 ll of 9 mM acetyl CoA (Sigma-Aldrich), with the reaction being carried out according to manufacturer’s instructions. Samples were then spotted at the bottom of a silica gel TLC plate (EM Science), allowed to air dry and finally placed in a closed

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glass chromatography tank containing a mixture of chloroform:methanol (9:1, v/v). After ascension of the solvent, the plate was removed from the tank to air-dry, and products were visualized under UV illumination. The rescue methodology of the minigenomes is illustrated in Figure 2A. Successful rescue results were only obtained with the minigenome plasmids containing the CAT reporter gene (Table 1). The best results were obtained when using expression plasmids containing a slightly weaker promoter to express the ANDV proteins or minigenome (CMV and T7 promoters) compared to plasmids with higher expression levels (CAG), although rescue was successful with all of the different constructs and CAT minigenome plasmids (Table 1). Numerous attempts were made to optimize the correct ratio of expression and minigenome plasmids in order to attain the proper biological conditions. Lower amounts of minigenome plasmids helped reduce background reporter signal levels that initially made interpretation of results difficult. Rescue attempts were also made using superinfection with ANDV in order to provide the necessary helper proteins, but these unfortunately also failed. The addition of a plasmid expressing the ANDV glycoproteins (GN and GC) did not have a noticeable effect on rescue efficacy either (data not shown). The minigenome cassettes were inserted in the vRNA orientation because they can be directly transcribed by the viral RNP complex to produce the reporter gene signal, whereas those in the cRNA orientation must first undergo a round of replication to produce the vRNA transcripts. Previously described minigenomes for Ebola and Marburg viruses used the vRNA orientation and showed a higher reporter signal compared to constructs in the cRNA orientation [9, 21]. The M-segment NCRs were chosen for the creation of the minigenome, as all members of the family Bunyaviridae contain the three genomic segments S, M and L, presumably with differences in relative promoter strength among them. Reports with the Uukuniemi virus minigenome suggested that the NCRs of the M segment contained the strongest promoter [7], whereas other studies have suggested the NCRs of the L segment may be stronger [6, 30]. One of the key aspects of successful minigenome rescue is to determine the appropriate biological conditions necessary for viral transcription and replication, through the variation of the ratio between the plasmids from which proteins forming the RNP complex and vRNA transcripts are expressed [2, 4]. A variety of different ratios of the N, L and minigenome plasmids were tried, using all of the different expression constructs described in this study in a variety of different cell lines, including Vero E6, 293T, HuH-7, A549 and COS-7. Expression of the CAT enzyme expressed from the minigenome containing the CAT reporter gene was successfully detected, indicating that functional L protein was

Andes virus minigenome

2231

A

B

C

con

-

-

+

D

+

con -

-

+

+

con -

-

-

+

MG

-

0.1

0.1

0.1

MG

-

1.0

1.0

0.3

MG

0.5

-

0.5

0.5

N

0.5

0.5

0.1

0.5

N

1.5

1.5

1.5

1.5

N

-

1.5

1.5

1.5

L

0.5

-

0.5

0.1

L

2.0

-

2.0

2.0

L

2.0

-

2.0

2.0

T7 Poly

1.0

1.0

1.0

1.0

T7 Poly

1.0

1.0

1.0

1.0

Fig. 2 Rescue of ANDV minigenome constructs. (A) Cartoon illustrating the minigenome rescue procedure. T7 bacteriophage (T7) RNA polymerase or human RNA polymerase I (Pol I) are used to transcribe the minigenome cassette containing the chloramphenicol acetyl transferase (CAT) reporter gene and M-segment non-coding regions (NCRs) in the viral RNA (vRNA) orientation. The addition of the ANDV polymerase (L) and nucleocapsid (N) proteins forms the ribonucleoprotein complex (RNP), which transcribes the minigenome in the messenger RNA orientation (mRNA). This transcript is translated into the functional CAT protein, which is detected by the CAT assay. (B) Rescue of the T7-based CAT minigenome using

CMV-driven RNP complex expression plasmids. (C) Rescue of the T7-based CAT minigenome using T7-driven RNP complex expression plasmids. (D) Rescue of the Pol-I-based CAT minigenome using CMV-driven RNP complex expression plasmids. Key: con = positive control plasmid containing the CAT gene expressed from the pCAGGS expression plasmid; - = negative rescue omitting either minigenome plasmid or one of the necessary RNP complex expression plasmids; ? = positive rescue indicated by CAT expression; N = ANDV nucleocapsid protein expression plasmid; L = ANDV RNA-dependent RNA polymerase expression plasmid; T7 poly = T7 RNA polymerase expression plasmid

being produced despite problems in the detection of L protein expression, although it remains unclear why the GFP and LUC mingenomes were not successfully rescued (Figure 2B-D). Despite numerous optimizations, the system continued to cause issues with reproducibility. The exact reason for this remains undetermined, but unreliable expression of the L protein is probably a major factor. Another explanation is the possible presence of a cryptic promoter in the NCRs, which has been described for other bunyaviruses [6, 7] or the presence of non-structural proteins that can influence

minigenome replication [1, 7, 11, 12, 16, 26, 27, 29]. Sequence analysis of the ANDV N protein ORF did identify two potential NSs proteins in different reading frames, one of which has very recently been identified as a native product during ANDV infection [28]. Expression plasmids for ANDV N with these potential ORFs knocked out through the modification of the ATG start codons were created (Table 1); however, the NSs knockout did not improve the minigenome rescue. The lack of reproducibility suggests that there is some hantavirus- or host-specific factor yet to be identified that appears to be interfering

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in the successful development of a reliable reverse genetics system. The successful development of systems for other members of the family Bunyaviridae suggests that the issue is not related to the tri-segmented RNA genome. The instability of L protein expression was a major issue in our system, and the successful production of a stable production of the L protein in conjunction with the N protein will be extremely important if a reliable system is to be achieved. Although stable production of the proteins is critical, obtaining the correct biological ratio of the proteins for virus rescue is also important, as mammalian expression vectors producing high levels of protein did not improve the rescue efficiency. Although minigenome systems for Hantaan virus, an Old World hantavirus, have been published [6, 30], our study presents the first minigenome system for a New World hantavirus. Despite these advances, a successful full-length infectious clone system has still not been established for any member of the genus Hantavirus, and the attempt described here for ANDV failed due to a lack of reproducibility and reliability in minigenome rescue. Acknowledgments This work funded by the National Microbiology Laboratory of the Public Health Agency of Canada. The work was part of the Ph.D. thesis of KSB. Thank you to Dr. Connie Schmaljohn, U.S Army Medical Research Institute of Infectious Diseases, Ft. Detrick, MD for providing the Andes virus, strain Chile 9717869.

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