10th international foamy virus conference 2014 · 2. high genetic variability of the envelope gene...

10th INTERNATIONAL FOAMY VIRUS CONFERENCE 2014

June 24‐25, 2014

National Veterinary Research Institute, Pulawy, Poland

Organized by: National Veterinary Research Institute Committee of Veterinary Sciences Polish Academy of Sciences Topics include: Foamy Virus Infection and Zoonotic Aspects, Foamy Virus Restriction and Immunity, Foamy Virus Biology and Gene Therapy Vectors

http://www.piwet.pulawy.pl/ifvc/

Organizers Jacek Kuźmak and Magdalena Materniak

Scientific Advisors

Martin Löchelt and Axel Rethwilm

Sponsors Support for the meeting was provided by the Committee of Veterinary Sciences Polish Academy of Sciences, ROCHE,

Thermo Fisher Scientific and EURx

Abstract book was issued thanks to the support provided by Committee of Veterinary Sciences Polish Academy of Sciences

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Program Overview

Monday, June 23rd, 2014

19 00 – 20 00 Registration

20 30 – open end Welcome Reception

Tuesday, June 24th, 2014


9 30 – 9 45 Welcome

9 45 – 10 15 Arifa Khan (FDA, Bethesda, US) - Simian foamy virus infection in natural host species

10 15 – 10 45 Antoine Gessain (Institute Pasteur, Paris, France) – Zoonotic human infection by

simian foamy viruses

10 45 – 11 20 Session 1 on Foamy Virus Infection and Zoonotic Aspects

Chairs: Arifa S. Khan and Antoine Gessain

11 20 – 11 45 Coffee break



12 30 – 13 00 Aris Katzourakis (Oxford University, UK - Mammalian genomes, their endogenous

viral elements, and the evolutionary history of the foamy viruses

13 00 – 14 15 Lunch Break

14 15 – 14 45 Dirk Werling (Royal Veterinary College, London, UK) - Messing up interferons – how

viruses impact on IFN signalling

14 45 – 15 45 Session 2 on Foamy Virus Restriction and Immunity

Chairs: Dirk Werling and Martin Löchelt


16 05 – 16 45 Rafal Kochanowski (ROCHE, Poland) – The future of laboratory developed testing –

The FLOW system powered by Roche Expand your potential today

18 00 Trip to Kazimierz Dolny and sightseeing 20 30 Dinner at Sarzyński Restaurant

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Wednesday, June 25th, 2014

9 30 – 10 10 Dirk Lindemann (TU Dresden, Germany) – Foamy Virus Structural protein

Biosynthesis and Particle egress: A Personal Perspective

10 10 – 11 10 Session 3 on Foamy Virus Replication, Proteins and Assembly

Chairs: Dirk Lindemann and Wentao Qiao

11 10 – 11 30 Coffee Break



14 15 – 14 50 Yahia Chebloune (University Joseph Fourier, Grenoble, France) – Retroviral vector

for gene transfer : are we ready to move on Foamy?

14 50 – 15 10 Session 4 on FV vectors

Chair: Yahia Chebloune

15 10 – 15 30 Arifa Khan (FDA, Bethesda, US) - FVs Taxonomy

15 30 – 16 30 Round-table Discussion on FVs Taxonomy and Prototype FV

Moderators: Arifa Khan, Martin Löchelt, Antoine Gessain, Aris Katzourakis and Dirk

Lindemann

16 30 Concluding Remarks

18 00 Barbecue in Janowiec castle

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Program

Monday, June 23rd, 2014


20 30 – open end Welcome Reception

Tuesday, June 24th, 2014


9 30 – 9 45 Welcome

9 45 – 10 15 Arifa Khan (FDA, Bethesda, US) - Simian foamy virus infection in natural host species

10 15 – 10 45 Antoine Gessain (Institute Pasteur, Paris, France) – Zoonotic human infection by

simian foamy viruses



Abst.#

1. Humans infected with SFV from chimpanzee and gorilla clades generate

neutralizing antibodies targeting antigenic determinants shared by both SFV clades

Caroline Lambert, Julie Gouzil, Réjane Rua, Edouard Betsem, Antoine Gessain and

Florence Buseyne

2. High genetic variability of the envelope gene of foamy viruses from Gorilla and

hunters infected by zoonotic strains

Léa Richard, Rejane Rua, Philippe V. Afonso, Edouard Betsem, Antoine Gessain




Abst.#

3. Screening for equine foamy virus infections in horses

Magdalena Materniak, Jerzy Rola, Wojciech Rożek, Aneta Pluta, Martin Löchelt, Jacek

Kuźmak

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4. The expression of bovine foamy virus in experimentally inoculated calves

Piotr Kubiś, Magdalena Materniak, Martin Löchelt, Jacek Kuźmak

5. Seroreactivity to non-primate foamy viruses in patients immunosupressed after

kidney transplantation

Magdalena Materniak, Agnieszka Serwacka, Andrzej Rydzewski, Sławomir Rudzki,

Łukasz Bocian, Timo Kehl, Martin Löchelt, Jacek Kuźmak

12 30 – 13 00 Aris Katzourakis (Oxford University, UK - Mammalian genomes, their endogenous

viral elements, and the evolutionary history of the foamy viruses


14 15 – 14 45 Dirk Werling (Royal Veterinary College, London, UK) - Messing up interferons –

how viruses impact on IFN signalling

14 45 – 15 45 Session 2 on Foamy Virus Restriction and Immunity


Abst. #

6. N-myc interactor inhibits prototype foamy virus by sequestering viral Tas protein

in the cytoplasm

Xiaomei Hu, Wei Yang, Ruikang Liu, Yunqi Geng Wentao Qiao, Juan Tan

7. Knockdown of the host cellular protein transportin 3 (TNPO3) attenuates

prototype foamy virus infection

Md. K. Ali, Faysal B. Hamid, Jin Sun Kim, Cha-Gyun Shin

8. Activation of epitope-specific CTLs using feline foamy virus vectors

Janet Lei, Wolfram Osen, Agnes Hotz-Wagenblatt, Stefan Eichmüller, Martin Löchelt


16 05 – 16 45 Rafal Kochanowski (ROCHE, Poland) – The future of laboratory developed testing –

The FLOW system powered by Roche Expand your potential today

18 00 Trip to Kazimierz Dolny and sightseeing

20 30 – open

end

Dinner at Sarzyński Restaurant

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Wednesday, June 25th, 2014

9 30 – 10 10 Dirk Lindemann (TU Dresden, Germany) – Foamy Virus Structural protein

Biosynthesis and Particle egress: A Personal Perspective



Abst.#

9. Nuclear trafficking of foamy virus

Joris Paris, Marie-Louise Giron, Antoine Bridier-Nahmias, Pascale Lesage, Philippe

Roingeard, Joëlle Tapiero-Tobaly, Ali Saïb

10. The cooperative function of arginine residues in the prototype foamy virus

Gag C-terminus mediates viral and cellular RNA encapsidation

Martin V. Hamann , Erik Müllers, Juliane Reh, Nicole Stanke, Dirk Lindemann

11. Importance of prototype-foamy virus Gag interaction with cellular polo-like

kinases

Irena Zurnic, Sylvia Hütter, Roger Helbig, Ute Lehmann, Gesche Gerresheim, Dirk

Lindemann

11 10 – 11 30 Coffee Break



12. The non-overlapping role of N-terminal residues of feline foamy virus Gag

during viral assembly and budding

Yang Liu, Matthew Betts, Janet Lei, Qiuying Bao, Guochao Wei, Axel Szabowski, Timo

Kehl, Robert Russell and Martin Löchelt

13. Molecular characterization of particle release phenotype of bovine foamy viruses

Qiuying Bao, Yang Liu, Michaela Hipp, Birgit Hub, Timo Kehl, Wentao Qiao and Martin

Löchelt

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14. Residue N108 of bovine foamy virus transactivator Tas contributes to its binding

with LTR and IP promoters

Tiejun Bing, Suzhen Zhang, Peng Shao, Song Zhang, Juan Tan, Wentao Qiao

15. Detection of highly expressed RNA polymerase III-directed retroviral

microRNAs of bovine foamy virus

Adam W. Whisnant, Timo Kehl, Qiuying Bao, Magdalena Materniak, Jacek Kuźmak,

Martin Löchelt and Bryan R. Cullen


14 15 – 14 50 Yahia Chebloune (University Joseph Fourier, Grenoble, France) – Retroviral vector

for gene transfer : are we ready to move on Foamy?

14 50 – 15 10 Session 4 on FV vectors


Abst. #

16. Foamy virus vectors for mesenchymal stem cell based gene therapy

Nathan P Sweeney,Cathy Regan, Jiahiu Liu, Begona Cachon Gonzalez, Dirk Lindemann,

Tony Rupar, Timothy Cox, Myra McClure

15 10 – 15 30 Arifa Khan (FDA, Bethesda, US) - talk on FVs Taxonomy

15 30 – 16 30 Round-table Discussion on FVs Taxonomy and Prototype FV (moderators: Arifa

Khan, Martin Löchelt, Antoine Gessain, Aris Katzourakis and Dirk Lindemann)

16 30 Concluding Remarks

18 00 - open end Barbecue in Janowiec castle

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Session 1

Foamy Virus Infection and Zoonotic Aspects


Abst.#

1. Humans infected with SFV from chimpanzee and gorilla clades generate neutralizing

antibodies targeting antigenic determinants shared by both SFV clades

Caroline Lambert, Julie Gouzil, Réjane Rua, Edouard Betsem, Antoine Gessain and Florence

Buseyne

2. High genetic variability of the envelope gene of foamy viruses from Gorilla and hunters

infected by zoonotic strains

Léa Richard, Rejane Rua, Philippe V. Afonso, Edouard Betsem, Antoine Gessain

3. Screening for equine foamy virus infections in horses

Magdalena Materniak, Jerzy Rola, Wojciech Rożek, Aneta Pluta, Martin Löchelt, Jacek

Kuźmak

4. The expression of bovine foamy virus in experimentally inoculated calves

Piotr Kubiś, Magdalena Materniak, Martin Löchelt, Jacek Kuźmak

5. Seroreactivity to non-primate foamy viruses in patients immunosupressed after kidney

transplantation

Magdalena Materniak, Agnieszka Serwacka, Andrzej Rydzewski, Sławomir Rudzki, Łukasz

Bocian, Timo Kehl, Martin Löchelt, Jacek Kuźmak

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Abstract 1 HUMANS INFECTED WITH SFV FROM CHIMPANZEE AND GORILLA

CLADES GENERATE NEUTRALIZING ANTIBODIES TARGETING ANTIGENIC DETERMINANTS SHARED BY BOTH SFV CLADES

Caroline Lambert a,b,c, Julie Gouzil a,b, Réjane Rua a,b, Edouard Betsem a,b, Antoine Gessain a,b and Florence Buseyne a,b

a Institut Pasteur, Unité d’épidémiologie et Physiopathologie des virus oncogènes, Institut Pasteur, Paris, France b CNRS, UMR3569, Paris, France c Université Paris Diderot, Cellule Pasteur, Paris, France Simian foamy viruses (SFVs) are efficiently transmitted from non-human primates to humans and may lead to chronic infection. Neither pathogenic effects nor human-to-human transmissions have been reported so far, supporting an efficient immune control of this retrovirus. Here, we aimed at studying the neutralizing antibodies present in the plasmas of SFV-infected persons living in rural areas in South Cameroun. Serial dilutions of plasma of 46 SFV-infected individuals and 7 uninfected persons were incubated with SFV for one hour; residual viral infectivity was quantified with the FAB (Foamy virus-activated beta-Gal expression) indicator cell line that expresses the beta-galactosidase gene under the control of the LTR from the SFVcpzPFV. Neutralization was assessed against three viral strains from the chimpanzee clade (SFVcpz): the SFVcpzPFV and SFVcpzSFV7 strains, which belong to serogroups 6 and 7 respectively, and the SFVcpzBAD327 strain, isolated from an individual from our study population. Plasmas from the 6 persons infected with SFVcpz neutralized either SFVcpzPFV or both SFVcpzSFV7 and SFVcpzBAD327. Plasmas from the 7 uninfected individuals and the 5 persons infected with SFV from monkeys did not neutralize any SFVcpz strain. Out of the 35 persons infected with SFV from the gorilla clade, 21 neutralized SFVcpzPFV only, 8 neutralized both SFVcpzSFV7 and SFVcpzBAD327, 1 neutralized the three strains, and 5 failed to neutralize any strain. Neutralizing titers ranged from 1/30 to 1/2060. Neutralizing titers against SFVcpzBad327 and SFVcpzSFV7 strains were strongly correlated (Spearman’s rho=0,9976, P<0,0001), supporting that the zoonotic SFVcpazBad327 strain belongs to serogroup 7. In conclusion, SFVs from the chimpanzee and gorilla clades transmitted to humans living in Cameroon share common antigenic properties and belong to at least 2 serogroups described in non-human primates. Plasmas with no neutralizing activities may belong to persons infected with viruses from a distinct serogroup or who may have failed to raise such immune response.

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Abstract 2 HIGH GENETIC VARIABILITY OF THE ENVELOPE GENE OF FOAMY VIRUSES FROM GORILLA AND HUNTERS INFECTED BY ZOONOTIC

STRAINS

Léa Richard a,b,c, Rejane Rua a,b,c, Philippe V. Afonso a,b, Edouard Betsem a,b,d, Antoine Gessain a,b

a Unit of Epidemiology and Physiopathology of Oncogenic Viruses, Department of Virology, Institut Pasteur, Paris, France b Centre National de la Recherche Scientifique (CNRS), UMR 3569, Paris, France c Université Paris Diderot, Cellule Pasteur, Paris, France d Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon

Simian Foamy Viruses (SFVs) are retroviruses believed to have co-evolved for millions of years with their hosts, the non-human primates (NHPs). This led to some variability between SFV strains. Moreover, co-infection with different strains of SFV has been shown in different NHP species; as a consequence, recombination between two strains (either from the same species or from different species) can occur, which expands further the retroviral diversity. In the framework of ongoing studies in South Cameroon, our goal is to study the genetic diversity of SFV strains isolated from African Apes and hunters infected with zoonotic strains. We have thus sequenced the SFV env gene (completely or partially) amplified from the blood DNA of: 1) a series of 30 humans infected with zoonotic SFV, originating mostly from gorilla and 2) a series of 5 gorillas or chimpanzees living in the same area of South Cameroon. The gorilla strains isolated from the infected individuals segregated into two distinct genetic variants that differed in a 750 bp long fragment of the env SU region. Interestingly, this region is similar to the one recently described as a recombinant “hot spot” region in the prototypic SFV-2 strain. One of the variant was present in a captive lowland gorilla and the other one in a wild-caught gorilla from Cameroon. Preliminary phylogenetic studies suggest that one variant derives from a recombination between a gorilla FV strain and another NHP strain, possibly of chimpanzee origin.

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Abstract 3

SCREENING FOR EQUINE FOAMY VIRUS INFECTIONS IN HORSES

Magdalena Materniak a, Jerzy Rola b, Wojciech Rożek b, Aneta Pluta a, Martin Löchelt c

and Jacek Kuźmak a a Department of Biochemistry, National Veterinary Research Institute, Pulawy, Poland b Department of Virology, National Veterinary Research Institute, Pulawy, Poland c Department of Genome Modifications and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany

Foamy viruses (FVs) are widely distributed among non-human primates, cats, cattle and horses. So far the least known is equine foamy virus (EFV), which was once reported to be isolated from blood of naturally infected horses. Its characteristic ultrastructure and ability to infect large number of cell lines make EFV similar to other foamy viruses, however its nucleotide sequence shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses little is known about epidemiology of EFV. Since our preliminary study suggested that EFV infections might be present among horses in Poland, we aimed to expand the diagnostics of EFV infections by developing specific diagnostic tools and apply them to investigate its prevalence. ELISA test based on recombinant EFV Gag protein was developed for serological investigation and qPCR for the detection of EFV DNA was established. 294 serum samples and 267 DNA samples from racing and saddle horses, Hucul ponies and semi-feral Polish primitive horses were subjected into the study. Due to the lack of positive and negative control samples, ELISA results were analysed statistically to determine the cut-off value. Based on the calculated cut off = 0,512 35 % of serum samples showed reactivity to EFV Gag protein. PCR detection was successful in 14.6% of DNA, however, there were some discordant results between both assays. Unfortunately all attempts to isolate EFV from leukocytes or lymphocytes of PCR positive horse blood samples failed. When the same sera were tested for the antibodies to equine arteritis virus (EAV), equine herpesvirus 1 (EHV 1), equine influenza virus A1 and A2 (EIV A1, EIV A2), statistically significant correlation (p < 0,03) was found between EFV and EIV A2 infections, while no correlation was noted between EFV and infections with other viruses. This is the first report confirming the existence of the infections with EFV in horses from Poland using virus-specific tools. This work was supported by grant no. 2011/01/B/NZ7/04282 from National Science Center.

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Abstract 4 THE EXPRESSION OF BOVINE FOAMY VIRUS IN EXPERIMENTALLY

INOCULATED CALVES

Piotr Kubiś a, Magdalena Materniak a, Martin Löchelt b, Jacek Kuźmak a a Department of Biochemistry, National Veterinary Research Institute, Puławy, Poland b Department of Genome Modifications and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany Bovine foamy virus is a member of Retroviridae family, Spumavirus genus and infects cattle under natural conditions. BFV is present in a high percentage of dairy cattle in different parts of the world. Transmission of BFV may occur through close contact pre- or perinatally, for instance, via colostrum or milk. In our previous studies we showed the presence of BFV DNA in different tissues of naturally and experimentally infected cows. However the primary site of virus replication has never been examined. The aim of this study was identification of BFV replication places and its level by one step RT-qPCR in experimentally inoculateded calves. In this study, five male calves of Holstein-Friesian breed (6 weeks old) seronegative for BFV, BLV and BVDV, were used. Three calves (calves # 6, 7, 9) were inoculated intravenously with 5 × 106 Cf2Th/BFV100 cells while two calves (calves # 8, 10) were inoculated with the same amount of uninfected Cf2Th cells as controls. Blood samples were taken from all animals 2, 3, 4, 12 and 20 weeks post inoculation (p. i.). Successful BFV infection was confirmed by qPCR in PBL’s of calves # 6, 7, 9 two weeks p. i. Total RNA was isolated from PBL’s to show the presence of viral RNA by RT-qPCR. It was detected in PBL’s of animal 7 2 and 3 weeks p. i., 12 weeks p. i. in PBL’s of animal 9 and in leukocytes of calves # 6, 7, 9 twenty weeks p. i. The highest number of copies of viral RNA/g of total RNA was 166.1 in leukocytes of calf 7 twenty weeks p.i. . RNA of BFV wasn’t detected in plasma samples of four calves # 6, 7, 8, 9 at 2 and 3 weeks p. i. BFV-inoculated and control calves were slaughtered 20 weeks p. i and internal organs such as spleen, lung, liver, kidney, testicle, tonsil, parotid, mesenteric and submaxillary lymph nodes, parotid and submaxillary salivary glands were taken. Also BAL cells and lamina propria lymphocytes were collected. Viral RNA was detected in spleen, lung, liver, parotid and mesenteric lymph nodes and BAL cells of all BFV-inoculated calves, in tonsil and submaxillary lymph node of calves # 7, 9 as well as in testicle of calf # 6. The highest number of copies of viral RNA/g of total RNA was 787.1 in parotid lymph node of calf # 9. No viral RNA was detected in thymus, kidney and salivary glands of BFV-inoculated animals nor in the PBL’s and tissues of control calves. Furthermore saliva samples of animals # 6, 7, 9 were collected twelve weeks p. i. and one week prior to slaughter and no viral RNA was detected. This is the first report showing that BFV replicates in vivo and indicating its possible replication sites.

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Abstract 5 SEROREACTIVITY TO NON-PRIMATE FOAMY VIRUSES IN PATIENTS

IMMUNOSUPPRESSED AFTER KIDNEY TRANSPLANTATION

Magdalena Materniak a, Agnieszka Serwacka b, Andrzej Rydzewski b, Sławomir Rudzki c, Łukasz Bocian d, Timo Kehl e, Martin Löchelt e and Jacek Kuźmak a

a Department of Biochemistry, National Veterinary Research Institute, Pulawy, Poland b Clinical Department of Internal Medicine, Nephrology and Transplantlogy of Central Clinical Hospital Ministry of Interior in Warsaw, Warsaw, Poland c First Chair and Department of General, Transplant Surgery and Clinical Nutrition, University of Medical Sciences, Lublin, Poland

d Department of Epidemiology and Risk Assessment, National Veterinary Research Institute, Pulawy, Poland

e Department of Genome Modifications and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany Interspecies transmissions of viruses are constant threat to human and animal health since they may result in new transmissible diseases with unpredictable pathogenic potential. Therefore the identification of suitable animal populations from which transmission to humans may occur and identification of human populations which are particularly at risk to be exposed to zoonotic agents or particularly vulnerable, such as immunocompromised individuals, is very important. It is now clearly demonstrated that foamy viruses can be zoonotically transmitted as was shown for men in Africa and Asia, exposed to contact with monkeys infected with simian foamy virus. But even though the exposure to foamy viruses to non-human primates is restricted to limited population of people, we can not exclude that exposure to FVs from companion and life-stock animals is much more likely and possibly affects very large population. Therefore we conducted serological studies of plasma samples collected from immunosuppressed patients using recombinant antigens specific for non-primate foamy viruses: bobvine foamy virus (BFV), feline foamy virus (FFV) and equine foamy virus (EFV). 117 blood samples were collected from immunosuppressed human beings, patients of the Clinic of Nephrology and Transplantlogy of Central Clinical Hospital in Warsaw. Another 44 blood samples were collected from control group, i.e. people donating blood for routine blood testing. All people were asked to fill in the questionnaire regarding their gender, age, place of residence, frequency and type of their contact with cows, horses and cats. Serum blood samples were tested for the presence of specific antibodies against EFV Gag, BFV Gag and Bet as well as FFV Gag and Bet antigens. In the group of immunosuppressed patients 2 samples showed increased reactivity to BFV antigens, 6 samples to FFV antigens and 8 to EFV antigen, while in control group 1 samples showed higher reactivity to BFV antigen, 2 to FFV antigens and 3 to EFV antigen. DNA samples corresponding to reactive sera were tested by PCR. Obtained results are subjected to statistical analysis. This study was supported by grant no. 2011/01/B/NZ7/04282 from National Science Center.

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http://www.cskmswia.pl/cse/medical-services/departments/9341,Clinical-Department-of-Internal-Medicine-Nephrology-and-Transplantlogy.html



Session 2

Foamy Virus Restriction and Immunity


Abst. #

6. N-myc interactor inhibits prototype foamy virus by sequestering viral Tas protein in the

cytoplasm


7. Knockdown of the host cellular protein transportin 3 (TNPO3) attenuates prototype

foamy virus infection

Md. K. Ali, Faysal B. Hamid, Jin Sun Kim, Cha-Gyun Shin

8. Activation of epitope-specific CTLs using feline foamy virus vectors

Janet Lei, Wolfram Osen, Agnes Hotz-Wagenblatt, Stefan Eichmüller, Martin Löchelt

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Abstract 6

N-MYC INTERACTOR INHIBITS PROTOTYPE FOAMY VIRUS BY SEQUESTERING VIRAL TAS PROTEIN IN THE CYTOPLASM


Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071, China

Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its deletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection.

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Abstract 7 KNOCKDOWN OF THE HOST CELLULAR PROTEIN TRANSPORTIN 3

(TNPO3) ATTENUATES PROTOTYPE FOAMY VIRUS INFECTION

Md. K. Ali, Faysal B. Hamid, Jin Sun Kim, Cha-Gyun Shin Department of Biotechnology,Chung Ang University, Ansung 456-75, Korea Transportin 3 (TNPO3) is a member of the importin-ß superfamily proteins. Despite numerous studies, the exact molecular mechanism of TNPO3 in retroviral infection is still controversial. Here, we provide evidence for the role and mechanism of TNPO3 in the replication of prototype foamy virus (PFV). Our findings revealed that PFV infection was reduced 2-fold by knockdown (KD) of TNPO3. However late stage of viral replication was not influenced. The differential cellular localization of PFV integrase (IN) in KD cells compared with control cells pinpointed a remarkable reduction of viral replication at the nuclear import step. We also found that TNPO3 interacted with PFV IN but not with Gag, suggesting that IN-TNPO3 interaction is important for nuclear import of PFV pre-integration complex. Our report enlightens the mechanism of PFV interaction with TNPO3 and support ongoing research on PFV as a promising safe vector for gene therapy.

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Abstract 8

ACTIVATION OF EPITOPE-SPECIFIC CTLs USING FELINE FOAMY VIRUS VECTORS

Janet Lei a, Wolfram Osen b, Agnes Hotz-Wagenblatt c, Stefan Eichmüller b, Martin Löchelt a a Department of Genome Modifications and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany b Group of Tumor Antigens, Department of Translational Immunology, German Cancer Research Center, Heidelberg, Germany c Bioinformatics Core Facility, German Cancer Research Center, Heidelberg, Germany Foamy viruses are apathogenic retroviuses from the Spumavirinae subfamily. With a broad cell tropism, high titers in vitro, and ability to efficiently transfer genes to target cells, they could be ideal vaccine scaffolds for long-term immune activation and stimulation. Using protein sequence alignment against a database of immune epitopes, we identified areas in feline foamy virus proteins that may allow epitope integration without disruption of essential viral functions and virus viability. The MHC-I epitope SIINFEKL, derived from ovalbumin, was used as a model antigen. Varying lengths of the epitope and its flanking regions were cloned into wild-type and replication-competent FFV and analyzed for proper protein expression, virion formation, replication competence, and long-term genetic stability. Results suggest that some modifications are better tolerated than others. We examined the ability of SIINFEKL-bearing Bet protein mutants and full viruses to present the epitope on the cell surface in the context of MHC-I. In an IFNγ ELISpot with anti-Ova cytotoxic T-lymphocytes (CTLs), subgenomic constructs bearing the SIINFEKL epitope significantly activated CTLs and promoted IFNγ release. Full virus bearing the epitope was also able, to a lower degree, to activate CTLs. Our results indicate that FFV and subgenomic constructs derived from FFV can be used as a T-cell epitope transfer vector for display on antigen-presenting cells.

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Session 3

Foamy Virus Replication, Proteins and Assembly


Abst. # 9. Nuclear trafficking of foamy virus

Joris Paris, Marie-Louise Giron, Antoine Bridier-Nahmias, Pascale Lesage, Philippe Roingeard,

Joëlle Tapiero-Tobaly, Ali Saïb

10. The cooperative function of arginine residues in the prototype foamy virus

Gag C-terminus mediates viral and cellular RNA encapsidation

Martin V. Hamann , Erik Müllers, Juliane Reh, Nicole Stanke, Dirk Lindemann

11. Importance of prototype-foamy virus Gag interaction with cellular polo-like kinases

Irena Zurnic, Sylvia Hütter, Roger Helbig, Ute Lehmann, Gesche Gerresheim, Dirk Lindemann

12. The non-overlapping role of N-terminal residues of feline foamy virus Gag during viral

assembly and budding

Yang Liu, Matthew Betts, Janet Lei, Qiuying Bao, Guochao Wei, Axel Szabowski, Timo Kehl,

Robert Russell and Martin Löchelt

13. Molecular characterization of particle release phenotype of bovine foamy viruses

Qiuying Bao, Yang Liu, Michaela Hipp, Birgit Hub, Timo Kehl, Wentao Qiao and Martin

Löchelt

14. Residue N108 of bovine foamy virus transactivator Tas contributes to its binding with

LTR and IP promoters

Tiejun Bing, Suzhen Zhang, Peng Shao, Song Zhang, Juan Tan, Wentao Qiao 15. Detection of highly expressed RNA polymerase III-directed retroviral microRNAs of

bovine foamy virus

Adam W. Whisnant, Timo Kehl, Qiuying Bao, Magdalena Materniak, Jacek Kuźmak, Martin

Löchelt and Bryan R. Cullen

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Abstract 9

NUCLEAR TRAFFICKING OF FOAMY VIRUS

Joris Paris a, Marie-Louise Giron a, Antoine Bridier-Nahmias a,c, Pascale Lesage a, Philippe Roingeard b, Joëlle Tapiero-Tobaly a, Ali Saïb a,c

a CNRS UMR7212, Inserm U944, Université Paris Diderot, Institut Universitaire d’Hématologie, Paris. b Université François Rabelais- Inserm E.A. 3856 - ERI 19, Tours c Conservatoire National des Arts et Métiers, Paris, France

The structural Gag protein hijacks many cellular machineries to fulfill its distinct and fundamental roles in the replication of retroviruses. In the case of the prototype foamy virus (PFV), Gag contains a nuclear export signal (NES) allows the protein to be exported back to the cytoplasm prior to capsids assembly and virus egress. We identified a nucleolar localization sequence (NoLS) in the C-terminus of PFV Gag. This NoLS contains two regions, rich in arginine and glycine, which are necessary and sufficient for nucleolar targeting. To study the nucleolar stage of PFV replication, we used conditions of hypoxia that slow down intracellular trafficking and also a molecular trap system to retain Gag into the nucleolus. In both cases, Gag was detected in the nucleolus. We also developed an approach, based on a ribozyme fused to a snoRNA (primary developed for HIV studies) and able to cleave specifically PFV RNA in the nucleolus, which allowed us to identify a nucleolar passage of PFV RNA. Although preliminary, our work suggests that PFV Gag might transit through the nucleolus to interact with the viral RNA and to export it from the nucleus. Importantly, this model predicts that the first capsid packaging unit could be made in the nucleolus. This role of Gag would be reminiscent of the role of Rev in HIV replication.

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Abstract 10

THE COOPERATIVE FUNCTION OF ARGININE RESIDUES IN THE PROTOTYPE FOAMY VIRUS GAG C-TERMINUS MEDIATES VIRAL

AND CELLULAR RNA ENCAPSIDATION

Martin V. Hamann a,b,c, Erik Müllers a,b,c, Juliane Reh a,b, Nicole Stanke a,b, Dirk Lindemann a,b

a Institute of Virology, Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, Dresden; b CRTD / DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Technische Universität Dresden, Dresden. c EM and MVH contributed equally

Foamy viruses (FVs) are a special type of retroviruses that combine replication characteristics of both orthoretroviruses and hepadnaviruses. One of several unique features of the FV capsid protein Gag is the absence of Cys-His motifs, which in orthoretroviruses are irreplaceable for multitude functions including viral RNA recognition and packaging. Instead, FV Gag contains glycine-arginine-rich (GR) sequences at its C-terminus. In case of prototype FV (PFV) these are grouped in three boxes, which have been shown to play essential functions in genome reverse transcription, virion infectivity and particle morphogenesis. Additional functions for RNA packaging and Pol encapsidation were suggested, although the contributions of individual boxes are controversially discussed. Here we show that the concurrent deletion of all three PFV Gag GR boxes or the substitution of 23 arginine residues residing in the C-terminal GR box region by alanine abolished both viral and cellular RNA encapsidation (>3,000-fold reduced), and in addition having also a negative effect on particle release (3 to 20-fold reduced). Those mutants lacked encapsidated Pol and were non-infectious. In contrast, deletion of individual GR boxes had only minor effects (2 to 4-fold) on viral and cellular RNA encapsidation over a wide range of cellular Gag to viral genome ratios examined. Taken together, our study provides the first description of cellular RNA encapsidation into FV particles and characterization of Gag mutants devoid of both viral and cellular RNA. Our results suggest that the cooperative action of C-terminal clustered positively charged residues is the main PFV Gag determinant for viral and cellular RNA encapsidation. This indicates that non-primate FVs, which lack clustering of Gag GR sequences in GR boxes, might use a similar mechanism of genome packaging. Furthermore, our work suggests that nucleic acid binding is a promoting, albeit not essential, mechanism for PFV particle assembly.

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Abstract 11

IMPORTANCE OF PROTOTYPE-FOAMY VIRUS GAG INTERACTION WITH CELLULAR POLO-LIKE KINASES

Irena Zurnic a,b, Sylvia Hütter a,b, Roger Helbig a,b, Ute Lehmann a,b, Gesche Gerresheim a,b, Dirk Lindemann a,b

a Institute of Virology, Medical Faculty “Carl Gustav Carus”, Technische Universität Dresden, Dresden, Germany b Center for Regenerative Therapies Dresden, Dresden, Germany Like all known viruses, prototype foamy virus (PFV) relies heavily on beneficial host machineries for successful replication. Previously, general steps of PFV entry, integration and egress have been described. However, little is known with respect to which cellular proteins aid viral replication via direct interaction with PFV structural components. To address this question, we performed a large yeast-two-hybrid (Y2H) screen using PFV Gag protein as a bait for identification of potential capsid interactants in mammalian cells. One candidate identified was human polo-like kinase 2 (hPlk2), a member of cell cycle regulatory kinases. In further Y2H studies, we confirmed interaction of PFV Gag with both human and rat Plk2 and identified an STP motif in Gag as essential for recognition by both kinases. Furthermore, we determined that functional kinase (KD) and polo-box domain (PBD) of Plk2 were required for direct interaction with the Gag protein. These results were corroborated by colocalization studies, where mCherry-tagged Gag colocalized with eGFP-tagged hPlk2 and rPlk2 at mitotic chromosomes in 293T cells ectopically expressing both proteins. Finally, the functionality of this interaction could be determined by assessing replication competent PFV particles bearing STP motif mutations for transduction efficiency. Whereas infectivity of these particles in one-round transduction experiments was decreased down to 20% of wild type, particle release and nucleic acid contents of all STP mutant-bearing particles were at wild type-like levels. Hence, we conclude that interaction between PFV Gag and Plk2 is important for early replication steps of PFV within a host cell. Gag mutants unable to interact with this polo-like kinase have diminished infectivity, but display wild type-like particle release, RNA packaging and reverse transcription efficiency. At which step exactly these particles are impeded in transduction remains to be determined by future analyses.

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Abstract 12

THE NON-OVERLAPPING ROLE OF N-TERMINAL RESIDUES OF FELINE FOAMY VIRUS GAG DURING VIRAL ASSEMBLY AND

BUDDING

Yang Liu a, Matthew Betts b, Janet Lei a, Qiuying Bao a, Guochao Wei a, Axel Szabowski a, Timo Kehl a, Robert Russell b and Martin Löchelt a

a Dept. Genome Modifications and Carcinogenesis, Research Program Infection and Cancer, German Cancer Research Center (DKFZ) b Structural Biology Group, Cell Networks Excellence Cluster, University of Heidelberg, Heidelberg, Germany The N-terminal region of Prototype Foamy Virus (PFV) Gag is responsible for particle budding via interaction with critical residues in Env/Elp. In contrast, the function of this region of Gag is ill-defined in the non-primate foamy viruses (FV) including feline FV (FFV). Mutagenesis of the N-terminus of FFV Gag reveals key residues essential for either capsid assembly and/or viral budding via an interaction with FFV Env leader protein (Elp). Sucrose gradient sedimentation analyses of cytosolic extracts of transfected cells clearly showed that wt Gag is mostly assembled into virus capsids and that un-assembled Gag and capsomeres of distinct size are undetectable. Proteolytic processing of Gag takes place only upon capsid assembly and in the presence of functional genomic RNA. This indicates that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. An appended heterogeneous myr signal rescues Gag particle budding but fails to generate particles that co-package Pol, as shown by a lack of Gag processing. In an in vitro Gag-Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Overall, the different phenotypic changes of these N-terminal Gag mutants, including proteolytic Gag processing, intracellular Gag assembly, and particle budding and infectivity, highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. The findings presented will be discussed based on a recent model developed for the PFV Gag – Elp interactions.

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Abstract 13

MOLECULAR CHARACTERIZATION OF PARTICLE RELEASE PHENOTYPE OF BOVINE FOAMY VIRUSES

Qiuying Bao a, Yang Liu a, Michaela Hipp a, Birgit Hub a, Timo Kehl a, Wentao Qiao b and Martin Löchelt a

a German Cancer Research Center, Focus Infection and Cancer, Heidelberg, Germany b Nankai University, Tianjin, China Bovine foamy virus (BFV) is a non-primate member of the Spumaretroviruses. Genomic analyses show close relationship to other foamy viruses (FVs). However, whereas the other FVs can be propagated in cell culture by cell-free infections, BFV is tightly cell-associated with an extremely inefficient transmission via cell-free virus particles. Consequently, the mechanism(s) responsible for the low level of cell-free BFV infectivity are currently investigated , which will be essential for better understanding the process of viral egress. For this purpose, variants with enhanced cell-free infectivity have been selected in parallel in two separate cultures by serially passaging using BICL (baby hamster kidney cells) and bovine MDBK (Madin-Darby bovine kidney epithelial cells) cells. BFV infectivity increased gradually and has plateaued at about 106 FFU/ml for BICl cells and 105 FFU/ml in MDBK cell. However, the BFV-specific cytopathic effect (CPE) was lost when increased titers are obtained. The complete gag and env genes have been cloned and sequenced from highly released BFV variants. Whereas consistent amino acid changes were detected in both genes, additional changes were apparently cell-type specific or stochastic. Since FV particle budding is Gag and Env-dependent, cotransfection of the selected genes displayed certain combinations with enhanced budding. In order to isolate Gag and Env sequences from the same parental virus with its specific co-evolution history, whole Gag-Pol-Env gene segments of BFV variants with high-level budding capacity were generated and analyzed. Consistent and cell-type specific genetic alterations were detected. Some clones showed a duplication of a potential budding-relevant L (late) domain. Most interestingly, specific and in-frame deletions in integrase from MDBK- and BICl-derived variants were detected. Consistent genetic changes in gag, pol and env will be studied in order to assess their contribution to the enhanced budding phenotype.

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Abstract 14

RESIDUE N108 OF BOVINE FOAMY VIRUS TRANSACTIVATOR TAS CONTRIBUTES TO ITS BINDING WITH LTR AND IP PROMOTERS

Tiejun Bing, Suzhen Zhang, Peng Shao, Song Zhang, Juan Tan, Wentao Qiao Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071, China Bovine foamy virus (BFV) encodes a transactivator called BTas that enhances viral gene transcription and is essential for BFV replication. BTas contains two major functional domains, the N-termianl DNA-binding domain (1-133 aa) and the C-terminal activation domain (198-249 aa), which are necessary for BTas transactivation activity. During the construction of BFV full-length genomic DNA clone (pBS-BFV), two clones (pBS-BFV-Y and pBS-BFV-B) with the same restriction enzyme pattern but different replication ability were identified. In this report, several chimeric clones were constructed, the replication activity assay showed that the viral C-terminal was more important for virus replication. Then the btas gene and LTR were subcloned into an eukaryotic expression plasmid and luciferase reporter plasmid, respectively. Transient transfection analyses showed that BTas-B encoded by pBS-BFV-B had higher transactivation ability than BTas-Y encoded by pBS-BFV-Y (~20 fold). Sequence alignment showed that there was only one aa different at position 108 between BTas-B (N108) and BTas-Y (D108), and then the important role of N108 was confirmed by mutagenesis analysis. In addition, we found the N108D mutation of BTas did not change its subcellular localization, homodimerization or the ability to activate NF-κB signal pathway, but could obviously enhance its binding ability to viral promoters both in vitro and in vivo. Interestingly, our preliminary results showed D108N could recover promoter binding and transactivation ability of BTas acetylation-deficient mutant, BTas-K66/109/110R. In addition, the N108D mutaion in pBS-BFV-B would reduce its replication ability to the same level of pBS-BFV-Y. Collectively, our findings suggest that N108 directly participates in BTas binding viral promoter and it is important for BFV replication.

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Abstract 15

DETECTION OF HIGHLY EXPRESSED RNA POLYMERASE III-DIRECTED RETROVIRAL microRNAs OF BOVINE FOAMY VIRUS

Adam W. Whisnant a, Timo Kehl b, Qiuying Bao b, Magdalena Materniak c, Jacek Kuźmak c, Martin Löchelt b and Bryan R. Cullen a

a Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina USA b German Cancer Research Center, Research Program Infection and Cancer, Heidelberg, Germany c Department of Biochemistry, National Veterinary Research Institute, Pulawy, Poland

MicroRNAs (miRNAs) are ~22-nt regulatory RNAs that are expressed by all known multicellular eukaryotes and several of their DNA viruses. miRNAs exert an additional layer of post-transcriptional control leading to a fine-tuning of cellular gene expression. In several DNA and few RNA viruses, miRNAs are essential e.g. for maintenance of virus persistence and latency or to promote replication. While numerous viral miRNAs expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Detection of miRNAs in bovine leukemia virus prompted us to search for miRNAs in bovine foamy virus (BFV), a member of the foamy virus sub-family of apparently non-pathogenic retroviruses that have potential as gene therapy and vaccine delivery vectors in human and veterinary medicine. The BFV miRNAs described here are unusual in that they are initially transcribed by RNA polymerase III as a single, ~122-nt long pri-miRNA. The Pol III transcript is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The three resultant mature miRNAs were found to contribute a remarkable ~70% of all miRNAs expressed in BFV-infected cells. The BFV miRNA cassette is located in the non-coding part of the BFV long terminal repeat “U3” region downstream of the accessory and regulatory bel genes. These data document the second example of a retrovirus that is able to express viral miRNAs using embedded proviral polymerase III promoters. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo. The data will be discussed with respect to foamy virus biology, for instance virus persistence in the host and the very long virus – host coevolution.

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Session 4

FV vectors


Abst. #

16. Foamy virus vectors for mesenchymal stem cell based gene therapy

Nathan P Sweeney,Cathy Regan, Jiahiu Liu, Begona Cachon Gonzalez, Dirk Lindemann, Tony

Rupar, Timothy Cox, Myra McClure

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Abstract 16 FOAMY VIRUS VECTORS FOR MESENCHYMAL STEM CELL BASED

GENE THERAPY

Nathan P Sweeney a,Cathy Regan b, Jiahiu Liu b, Begona Cachon Gonzalez c, Dirk Lindemann d, Tony Rupar b, Timothy Cox c, Myra McClure a

a Imperial College London, UK b University of Western Ontario, Canada c University of Cambridge, UK d Technische Universität Dresden, Germany Mesenchymal stem cells (MSCs) are a promising adult stem cell for use in regenerative medicine and have been shown to be safe in humans. In contrast to hematopoietic stem cells, the potential of combined stem cell and gene therapy approaches using MSCs has not been well evaluated. We have used foamy virus vectors to transduce MSCs with over 95% transduction efficiency. We propose and assess the potential of a novel approach of delivering foamy virus transduced MSCs directly to the brain as a therapy for neurodegenerative lysosomal storage diseases.

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CONFERENCE CONTACT LIST Qiuying Bao Department of Genome Modifications and Carcinogenesis German Cancer Research Center Heidelberg, Germany [email protected] Tiejun Bing Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) Key Laboratory of Microbial Functional Genomics (Tianjin) College of Life Sciences, Nankai University Tianjin, China [email protected] Florence Buseyne Institut Pasteur, Unité d'épidémiologie et physiopathologie des virus oncogènes Institut Pasteur, Paris, France [email protected] Yahia Chebloune Laboratoire Pathogénèse et Vaccination Lentivirales : PAVAL Lab. Université Joseph Fourier Grenoble 1, Pôle Chimie, Sciences du Vivant de la Santé et Bio-ingénierie, Grenoble Cedex 9, France [email protected] Antoine Gessain Institut Pasteur, Unité d'épidémiologie et physiopathologie des virus oncogènes Institut Pasteur Paris, France [email protected] Martin Hamann Institute of Virology, Medical Faculty "Carl Gustav Carus" Technical University Dresden, Germany [email protected]

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mailto:[email protected]






Xiaomei Hu Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) Key Laboratory of Microbial Functional Genomics (Tianjin) College of Life Sciences, Nankai University Tianjin, China [email protected] Aris Katzourakis Department of Zoology Oxford Univeristy Oxford, UK [email protected] Arifa S. Khan Laboratory Of Retroviruses Division of Viral Products OVRR, CBER U.S. Food and Drug Administration Bethesda, USA [email protected] Piotr Kubiś Department of Biochemistry National Veterinary Research Institute Pulawy, Poland [email protected] Jacek Kuźmak Department of Biochemistry National Veterinary Research Institute Pulawy, Poland [email protected] Caroline Lambert Institut Pasteur, Unité d'épidémiologie et physiopathologie des virus oncogènes Institut Pasteur Paris, France [email protected]

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Janet Lei Department of Genome Modifications and Carcinogenesis German Cancer Research Center Heidelberg, Germany [email protected] Dirk Lindemann Institute of Virology, Medical Faculty "Carl Gustav Carus" Technical University Dresden, Germany [email protected] Martin Löchelt Department of Genome Modifications and Carcinogenesis German Cancer Research Center Heidelberg, Germany [email protected] Magdalena Materniak Department of Biochemistry National Veterinary Research Institute Pulawy, Poland [email protected] Joris Paris CNRS UMR7212, Inserm U944 Université Paris Diderot Institut Universitaire d’Hématologie Paris, France [email protected] Wentao Qiao Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) Key Laboratory of Microbial Functional Genomics (Tianjin) College of Life Sciences, Nankai University Tianjin, China [email protected]

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Axel Rethwilm Institut für Virologie und Immunbiologie Universität Würzburg Würzburg, Germany [email protected] Léa Richard Institut Pasteur, Unité d'épidémiologie et physiopathologie des virus oncogènes Institut Pasteur Paris, France [email protected] Marzena Rola-Łuszczak Department of Biochemistry National Veterinary Research Institute Pulawy, Poland [email protected] Cha-Gyun Shin Department of Biotechnology Chung-Ang University Ansung, South Korea [email protected] Nathan Sweeney Infectious Diseases Imperial College London London, United Kingdom [email protected] Axel Szabowski Department of Genome Modifications and Carcinogenesis German Cancer Research Center Heidelberg, Germany [email protected]

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Guochao Wei Department of Genome Modifications and Carcinogenesis German Cancer Research Center Heidelberg, Germany [email protected] Dirk Werling Pathology and Pathogen Biology The Royal Veterinary College Hatfield, Herts, United Kingdom [email protected] Irena Zurnic Institute of Virology, Medical Faculty "Carl Gustav Carus" Technical University Dresden, Germany [email protected]




10th international foamy virus conference 2014 · 2. high genetic variability of the envelope gene...

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