Primate foamy virus Pol proteins are imported into the nucleus

Horst Imrich1, Martin Heinkelein1, Ottmar Herchenröder2 and Axel Rethwilm1,2

Institut für Virologie und Immunbiologie, Universität Würzburg, Germany1
Institut für Virologie im MTZ, Medizinische Fakultät ‘Carl Gustav Carus’, Technische Universität Dresden, Fiedlerstr. 42, 01307 Dresden, Germany2

Author for correspondence: Axel Rethwilm (at the Technische Universität Dresden). Fax +49 351 458 6314. e-mail Axel.Rethwilm{at}mailbox.tu-dresden.de


   Abstract
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Abstract
Introduction
References
 
Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127Pol, p85RT/RN and p40IN served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.


   Introduction
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Abstract
Introduction
References
 
Foamy viruses make up a distinct group of reverse transcriptase-utilizing viruses that use a replication strategy different from those of retroviruses and hepadnaviruses (Linial, 1999 ). The best-studied foamy virus is the so-called human foamy virus (HFV). However, a true human foamy virus does not exist (Linial, 2000 ); what does exist are zoonotic human infections by non-human primate foamy viruses (PFVs). Therefore, we have adopted the term PFV instead of HFV to indicate this. The nuclear localization of PFV Gag protein is one of the remarkable features that separate these viruses from other retroviruses (Hooks & Gibbs, 1975 ; Schliephake & Rethwilm, 1994 ). It has been shown previously that newly synthesized Gag is actively transported into the nucleus of PFV-infected cells due to a nuclear localization signal (NLS) situated in the C-terminal part of Gag (the GRII box) (Schliephake & Rethwilm, 1994 ). The reason for this nuclear accumulation of PFV Gag protein during the virus replication cycle is not yet known. However, the replication competence of a partial GRII box deletion mutant, which was reported to show little nuclear Gag localization, indicated that the nuclear transit of Gag may not be essential for PFV replication in vitro (Yu et al., 1996a ). In addition to Gag, nuclear localization of Pol protein has been reported in PFV-infected cells by using polyclonal rabbit antisera in immunoblot assays following cell fractionation (Morozov et al., 1997 ). PFV Pol is expressed independently of Gag from a spliced pol mRNA (Enssle et al., 1996 ; Jordan et al., 1996 ; Yu et al., 1996b ). This mode of expression means that Pol is not transported into the nucleus as part of a Gag–Pol fusion protein. However, whether the PFV Pol protein is co-imported into the nucleus via non-covalent linkage to Gag or whether it can also enter the nucleus independently has not been investigated. Moreover, the time-course of the Pol nuclear pathway remains to be determined. The PFV 127 kDa Pol precursor protein is cleaved into at least two subunits: the approx. 85 kDa protease/reverse transcriptase/RNase H (PR/RT/RN) and the approx. 40 kDa integrase (IN) (Netzer et al., 1990 ; Pfrepper et al., 1998 ). Therefore, we were also interested to find out which of the three molecules is actually localized to the nucleus of infected cells. To investigate these topics, we generated MAbs directed against both Pol subunits to be used in immunofluorescence assays (IFA).

C57BL/6 mice (Harlan & Winkelmann) were infected with 106 cell-free infectious units of PFV derived from cells transfected with the pHSRV2 molecular clone (Schmidt & Rethwilm, 1995 ) via the intraperitoneal (i.p.) route as described previously (Schmidt et al., 1997 ). Six months after infection, strong humoral responses against Gag and Bet proteins were detected in the serum of infected mice (data not shown). The mice were superinfected i.p. with 5x107 p.f.u. of a recombinant vaccinia virus (VV) that expressed PFV Pol protein (VVpol1; Fischer et al., 1997 ). Two months after superinfection, the antibody response against Pol was further boosted by immunization with bacterially expressed protein. To generate this, the entire PFV pol open reading frame was cloned into the pET15b vector (Novagen). Following induction of protein expression in E. coli BL21(DE3), the recombinant protein was purified via an N-terminal histidine tag by affinity chromatography on a Ni2+ chelation column as directed by the supplier (Novagen). Fifty µg of purified antigen in 100 µl incomplete Freund’s adjuvant was used to immunize mice by the i.p. route. Two weeks after administration of this booster, the mice were sacrificed and 1x108 spleen cells were fused with 4x107 Sp2/0-Ag14 cells (Shulman et al., 1978 ) and selected in HAT and HT medium (Sigma) by using standard protocols (Harlow & Lane, 1988 ). The supernatants were screened by ELISA on microtitre plates coated with the purified bacterially expressed Pol antigen by using alkaline phosphatase-conjugated goat anti-mouse antibody (DAKO). Antibody-positive supernatants were analysed further in immunoblots with lysates of PFV-infected cells as described previously (Hahn et al., 1994 ). Three cell clones secreted antibodies stably following five limiting-dilution subcloning steps. All MAbs were determined to be of the IgG2a subtype (Coligan et al., 1994 ). The hybridomas designated 8A3 and 15E10 recognize pr127Pol and p85RT/RN and clone 3E11 reacts with pr127Pol and p40IN (Fig. 1A). In addition, MAb 3E11 specifically detects a viral protein with an apparent molecular mass of approx. 70 kDa in PFV-infected baby hamster kidney cells (BHK-21), but not in uninfected cells (Fig. 1A). As an additional control for specificity, we used lysates of 3T3 TK- cells infected with two VV recombinants that express either wild-type PFV Pol protein (VVpol1) or a Pol protein bearing a mutation in the active site of the protease (VVpol2), which is therefore unable to cleave the 127 kDa precursor into subunits (Konvalinka et al., 1995 ; Fischer et al., 1997 ). As shown in Fig. 1(A), the respective PFV proteins were detected in these lysates by MAbs 3E11 and 15E10. The 70 kDa protein was also recognized by MAb 3E11 in lysates from VVpol1- and VVpol2-infected cells, which demonstrated that an active viral protease was not required to generate this Pol-related protein. The characteristics of this protein are currently not known.



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Fig. 1. Reactivity of Pol-specific MAbs with individual Pol protein subunits and depiction of eukaryotic pol expression constructs. (A) Immunoblot of lysates from PFV-infected (HSRV2 isolate) BHK-21 cells, from 3T3 TK- cells infected with recombinant VV expressing a wild-type PFV polymerase (VVpol1) or a polymerase with mutated protease active site (VVpol2) and from 293T cells transfected with the individual pol expression constructs shown in (B). The lysates were prepared from uninfected or infected cells and the proteins were separated in SDS–PAGE gels and transferred to nitrocellulose as described previously (Hahn et al., 1994 ). Filters were incubated with supernatants from the MAb hybridoma cultures and, after reaction with peroxidase-labelled second antibody, were developed by using the ECL-detection system (Amersham). MAbs 8A3 and 15E10 detected the pr127Pol precursor protein and p85RT/RN specifically in PFV-infected cells. MAb 15E10 was also analysed with lysates prepared from recombinant VV-infected cells. While VVpol1 expresses pr127Pol and p85RT/IN, VVpol2 can only express pr127Pol due to inactivation of the pol-encoded protease (Fischer et al., 1997 ). MAb 3E11 detected pr127Pol and p40IN. In addition, this antibody reacted with a viral protein of approx. 70 kDa in wild-type infected cells and in cells infected with the recombinant VV. The nature of this protein is currently unknown. In lysates from transfected 293T cells, the antibodies specifically detected the precursor molecule, cleaved subunits and proteins expressed from the subgenic plasmids. The latter were of similar apparent sizes as the subunits cleaved from the precursor. (B) Constructs directing the eukaryotic expression of Pol proteins. All constructs are in a pcDNA vector backbone (Invitrogen). pcPol2 has been described previously (Heinkelein et al., 1998 ). pcRT2 and pcIN1 were generated by recombinant PCR (Higuchi, 1990 ), introducing translational stop signals at the predicted RT/RN–IN cleavage site (Pfrepper et al., 1998 ) or fusing the IN reading frame downstream of the cleavage site to the pol ATG start codon, respectively. The polyadenylation signal (pA+) was derived from the pcDNA vector.

 
BHK-21 cells were infected with PFV at an m.o.i. of 10 for 2 h on ice. The cells were then washed and seeded at a density of 3x105 onto coverslips in 6 cm Petri dishes and fixed in cold methanol after incubation periods ranging from 24 to 96 h. Double IFA staining was performed with rabbit anti-Gag serum and FITC-labelled goat anti-rabbit second antibody and with the mouse MAbs and Texas red-labelled goat anti-mouse second antibody (second antibodies were from Jackson Immuno Research) as described previously (Schliephake & Rethwilm, 1994 ). As shown in Fig. 2(A, B), Gag and Pol proteins were mainly detected in the cytoplasm of infected cells at 24 h after infection. At 48 h after infection, most of the anti-Gag stain was detected in the nucleus (green fluorescence), while Pol proteins were detected in the cytoplasm (red fluorescence) and in the nuclei, as revealed by the overlapping yellow fluorescence (Fig. 2C, D). Subsequently, until disintegration of the cells by PFV-induced CPE, Gag protein was detected almost exclusively in the nuclei of infected cells while the anti-Pol stain showed a predominant nuclear fluorescence, and only a small amount of protein was located in the cytoplasm (Fig. 2E, F). The nuclear localization of Pol proteins was also revealed by single IFA, as shown in Fig. 2(G, H). Although we observed some heterogeneity of staining in individual experiments late in infection, there was no significant difference in subcellular distribution of Pol when using the MAbs that recognized different domains of the polyprotein. This and the co-localization with Gag raised the question of whether nuclear transfer of Pol proteins occurs exclusively in a complex with Gag protein or whether they are also translocated to the nucleus independently of Gag.



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Fig. 2. IFA of BHK-21 cells infected with PFV at an m.o.i. of 10 with Gag-specific rabbit antiserum and Pol-specific MAbs at 24 (A, B), 48 (C, D) and 72 (E–H) h post-infection. Samples shown in (A)–(F) were stained with a rabbit polyclonal antiserum generated against PFV capsid protein expressed in the baculovirus system (Hahn et al., 1994 ) and FITC-conjugated second antibody. Samples shown in (A), (C), (E) and (G) were stained with IN-specific MAb 3E11, while those in (B), (D), (F) and (H) were stained with RT/RN-specific MAb 15E10 with Texas red-coupled second antibody.

 
In order to analyse this, we generated eukaryotic expression plasmids for pr127Pol (pcPol2), p85RT/RN (pcRT2) and p40IN (pcIN1), as shown in Fig. 1(B), by standard recombinant DNA techniques (Ausubel et al., 1987 ). 293T cells were transfected with the plasmids as described previously and analysed by immunoblot and IFA (Heinkelein et al., 2000b ). In immunoblot, 15E10 and 3E11 specifically detected bands of 85 and 40 kDa in lysates from pcRT2- and pcIN1-transfected cells, respectively (Fig. 1A).

IFA of 3T3 TK- cells infected with VVpol1 and VVpol2 using RT/RN-specific MAb 15E10 (Fig. 3A, B) or IN-specific MAb 3E11 (Fig. 3C, D) showed some cytoplasmic staining, but mainly nuclear fluorescence. A similar picture emerged when 293T cells transfected with pcPol2 (Fig. 3E, G), pcRT2 (Fig. 3F) or pcIN1 (Fig. 3H) were incubated with 15E10 (Fig. 3E, F) or 3E11 (Fig. 3G, H). Counterstaining of nuclei with Hoechst 33342 revealed that both the p85RT/RN and p40IN subunits of the Pol precursor are mainly nuclear proteins, irrespective of whether they were expressed together or individually.



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Fig. 3. Nuclear fluorescence of 3T3 TK- cells infected with recombinant VV and 293T cells transfected with subgenic pol expression constructs. (A)–(D) IFA of 3T3 TK- cells infected with VVpol1 (A, C) or VVpol2 (B, D) with MAbs 15E10 (A, B) or 3E11 (C, D). (E)–(H) IFA of 293T cells transfected with pcPol2 (E, G), pcRT2 (F) or pcIN1 (H) with MAbs 15E10 (E, F) or 3E11 (G, H). The nuclei of 293T cells were counterstained with Hoechst 33342. All reactions were performed with Texas red-coupled goat anti-mouse serum.

 
In retroviruses, nuclear localization of IN is necessary in order to establish a new provirus by insertion of the long terminal repeat (LTR)-flanked viral genome into cellular DNA (Mumm et al., 1992 ; Gallay et al., 1997 ; Petit et al., 1999 ; Pluymers et al., 1999 ; Kukolj et al., 1997 , 1998 ). As revealed by immunoblot analysis, at most 50% of pr127 Pol molecules are cleaved intracellularly to form the p85RT/RN and p40IN subunits (Fig. 1A). Nuclear localization of the pr127 molecule would be expected because of the proposed IN NLS. Surprisingly, in addition, we found that exclusive expression of the RT/RN domain also resulted in nuclear accumulation (Fig. 3). Both pr127Pol and p85RT/RN are too large to cross the nuclear membrane passively (Garcia-Bustos et al., 1991 ). Thus, we propose that PFV possesses at least two NLS in the Pol polyprotein, one of which is located in the RT/RN domain and the other in the IN domain.

It is interesting to note that PFV capsids therefore appear to bear three separate NLS distributed to both the Gag and Pol proteins. However, the function of these redundant signals is still not known. Human immunodeficiency virus (HIV) has separate NLS in the Gag, IN and Vpr proteins for the nuclear import of the pre-integration complex, which is believed to play a crucial role upon infection of resting cells (Gallay et al., 1997 ; Popov et al., 1998 ). In contrast, PFVs do not infect resting cells efficiently and IFA with a Gag antibody failed to detect the PFV pre-integration complex in the nucleus shortly after infection (Bieniasz et al., 1995 ; Saïb et al., 1997 ). Furthermore, the replication strategy of PFV deviates from HIV and all other retroviruses (Rethwilm, 1996 ; Linial, 1999 ). The most remarkable features of this distinct PFV replication pathway are reverse transcription occurring late in replication and the accumulation of proviral integrants via intracellular retrotransposition (Moebes et al., 1997 ; Heinkelein et al., 2000a ). Multiple integration has also been described in H92 erythroleukaemia cells persistently infected with PFV, with the number of integrants depending on the presence of a fully functional Gag NLS (Meiering et al., 2000 ). Although an extracellular mechanism of provirus acquisition could not be excluded in the latter study, we would like to suggest that the presence of staggered NLS in proteins constituting PFV cores are required for an internal replication cycle, as similarly suggested for hepatitis B virus (Nassal & Schaller, 1993 ). This replicative short cut would result in the accumulation of nuclear viral DNA forms rather than further spread within the host by the generation of infectious cell-free virus.

PFV can be readily isolated from various host tissues, although virus replication in vivo appears to be very limited (Falcone et al., 1999 ). Upon propagation in vitro, PFVs give rise to a remarkable CPE of host cells characterized by the formation of large syncytia and eventually leading to cellular disintegration. Perhaps the proposed intracellular replication pathway may explain the life-long benign persistence of PFV.


   Acknowledgments
 
This work was supported by grants from the EU (BMH4-CT97-2010), BMBF (01KV9817/0), DFG (Re627/6-1 and SFB479), Sächsisches Staatsministerium für Umwelt und Landwirtschaft, Medizinische Fakultät ‘Carl Gustav Carus’ and Bayerische Forschungsstiftung (Forgen).


   References
Top
Abstract
Introduction
References
 
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1987). Current Protocols in Molecular Biology. New York: John Wiley.

Bieniasz, P. D., Weiss, R. A. & McClure, M. O.(1995). Cell cycle dependence of foamy retrovirus infection.Journal of Virology69, 7295-7299.[Abstract]

Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. & Strober, W. (1994). Current Protocols in Immunology. New York: John Wiley.

Enssle, J., Jordan, I., Mauer, B. & Rethwilm, A.(1996). Foamy virus reverse transcriptase is expressed independently from the Gag protein.Proceedings of the National Academy of Sciences, USA93, 4137-4141.[Abstract/Free Full Text]

Falcone, V., Leupold, J., Clotten, J., Urbanyi, E., Herchenröder, O., Spatz, W., Volk, B., Böhm, N., Toniolo, A., Neumann-Haefelin, D. & Schweizer, M.(1999). Sites of simian foamy virus persistence in naturally infected African green monkeys: latent provirus is ubiquitous, whereas viral replication is restricted to the oral mucosa.Virology257, 7-14.[Medline]

Fischer, N., Enssle, J., Müller, J., Rethwilm, A. & Niewiesk, S.(1997). Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses. AIDS Research and Human Retroviruses 13, 517-521.[Medline]

Gallay, P., Hope, T., Chin, D. & Trono, D.(1997). HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway.Proceedings of the National Academy of Sciences, USA94, 9825-9830.[Abstract/Free Full Text]

Garcia-Bustos, J., Heitman, J. & Hall, M. N.(1991). Nuclear protein localization.Biochimica et Biophysica Acta1071, 83-101.[Medline]

Hahn, H., Baunach, G., Bräutigam, S., Mergia, A., Neumann-Haefelin, D., Daniel, M. D., McClure, M. O. & Rethwilm, A.(1994). Reactivity of primate sera to foamy virus Gag and Bet proteins.Journal of General Virology75, 2635-2644.[Abstract]

Harlow, E. & Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Heinkelein, M., Schmidt, M., Fischer, N., Moebes, A., Lindemann, D., Enssle, J. & Rethwilm, A.(1998). Characterization of a cis-acting sequence in the Pol region required to transfer human foamy virus vectors.Journal of Virology72, 6307-6314.[Abstract/Free Full Text]

Heinkelein, M., Pietschmann, T., Jarmy, G., Dressler, M., Imrich, H., Thurow, J., Lindemann, D., Bock, M., Moebes, A., Roy, J., Herchenröder, O. & Rethwilm, A.(2000a). Efficient intracellular retrotransposition of an exogenous primate retrovirus genome. EMBO Journal19, 3436-3445.[Abstract/Free Full Text]

Heinkelein, M., Thurow, J., Dressler, M., Imrich, H., Neumann-Haefelin, D., McClure, M. O. & Rethwilm, A.(2000b). Complex effects of deletions in the 5’ untranslated region of primate foamy virus on viral gene expression and RNA packaging.Journal of Virology74, 3141-3148.[Abstract/Free Full Text]

Higuchi, R. (1990). Recombinant PCR. In PCR Protocols: A Guide to Methods & Applications, pp. 177–183. Edited by M. A. Innis, D. H. Gelfand & T. J. White. San Diego, CA: Academic Press.

Hooks, J. J. & Gibbs, C. J.Jr(1975). The foamy viruses.Bacteriological Reviews39, 169-185.[Medline]

Jordan, I., Enssle, J., Güttler, E., Mauer, B. & Rethwilm, A.(1996). Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA.Virology224, 314-319.[Medline]

Konvalinka, J., Löchelt, M., Zentgraf, H., Flügel, R. M. & Kräusslich, H.-G.(1995). Active foamy virus proteinase is essential for virus infectivity but not for formation of a Pol polyprotein.Journal of Virology69, 7264-7268.[Abstract]

Kukolj, G., Jones, K. S. & Skalka, A. M.(1997). Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases.Journal of Virology71, 843-847.[Abstract]

Kukolj, G., Katz, R. A. & Skalka, A. M.(1998). Characterization of the nuclear localization signal of the avian sarcoma virus integrase. Gene223, 157-163.[Medline]

Linial, M. L.(1999). Foamy viruses are unconventional retroviruses.Journal of Virology73, 1747-1755.[Free Full Text]

Linial, M. L.(2000). Why aren’t foamy viruses pathogenic?Trends in Microbiology8, 284-289.[Medline]

Meiering, C. D., Comstock, K. E. & Linial, M. L.(2000). Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells.Journal of Virology74, 1718-1726.[Abstract/Free Full Text]

Moebes, A., Enssle, J., Bieniasz, P. D., Heinkelein, M., Lindemann, D., Bock, M., McClure, M. O. & Rethwilm, A.(1997). Human foamy virus reverse transcription that occurs late in the viral replication cycle. Journal of Virology71, 7305-7311.[Abstract]

Morozov, V. A., Copeland, T. D., Nagashima, K., Gonda, M. A. & Oroszlan, S.(1997). Protein composition and morphology of human foamy virus intracellular cores and extracellular particles. Virology228, 307-317.[Medline]

Mumm, S. R., Hippenmeyer, P. J. & Grandgenett, D. P.(1992). Characterization of a stable eukaryotic cell line expressing the Rous sarcoma virus integrase.Virology189, 500-510.[Medline]

Nassal, M. & Schaller, H.(1993). Hepatitis B virus replication.Trends in Microbiology1, 221-228.[Medline]

Netzer, K.-O., Rethwilm, A., Maurer, B. & ter Meulen, V.(1990). Identification of the major immunogenic structural proteins of human foamy virus.Journal of General Virology71, 1237-1241.[Abstract]

Petit, C., Schwartz, O. & Mammano, F.(1999). Oligomerization within virions and subcellular localization of human immunodeficiency virus type 1 integrase.Journal of Virology73, 5079-5088.[Abstract/Free Full Text]

Pfrepper, K.-I., Rackwitz, H.-R., Schnölzer, M., Heid, H., Löchelt, M. & Flügel, R. M.(1998). Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease.Journal of Virology72, 7648-7652.[Abstract/Free Full Text]

Pluymers, W., Cherepanov, P., Schols, D., De Clercq, E. & Debyser, Z.(1999). Nuclear localization of human immunodeficiency virus type 1 integrase expressed as a fusion protein with green fluorescent protein.Virology258, 327-332.[Medline]

Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, M.-A., Ratner, L., Lane, C. M., Moore, M. S., Blobel, G. & Bukrinsky, M.(1998). Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.EMBO Journal17, 909-917.[Abstract/Free Full Text]

Rethwilm, A. (1996). Unexpected replication pathways of foamy viruses. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 13 (Suppl. 1), S248–S253.[Medline]

Saïb, A., Puvion-Dutilleul, F., Schmid, M., Périès, J. & de Thé, H.(1997). Nuclear targeting of incoming human foamy virus Gag proteins involves a centriolar step.Journal of Virology71, 1155-1161.[Abstract]

Schliephake, A. W. & Rethwilm, A.(1994). Nuclear localization of foamy virus Gag precursor protein.Journal of Virology68, 4946-4954.[Abstract]

Schmidt, M. & Rethwilm, A.(1995). Replicating foamy virus-based vectors directing high level expression of foreign genes. Virology210, 167-178.[Medline]

Schmidt, M., Niewiesk, S., Heeney, J., Aguzzi, A. & Rethwilm, A.(1997). Mouse model to study the replication of primate foamy viruses.Journal of General Virology78, 1929-1933.[Abstract]

Shulman, M., Wilde, C. D. & Köhler, G.(1978). A better cell line for making hybridomas secreting specific antibodies. Nature276, 269-270.[Medline]

Yu, S. F., Edelmann, K., Strong, R. K., Moebes, A., Rethwilm, A. & Linial, M. L.(1996a). The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.Journal of Virology70, 8255-8262.[Abstract]

Yu, S. F., Baldwin, D. N., Gwynn, S. R., Yendapalli, S. & Linial, M. L.(1996b). Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses.Science271, 1579-1582.[Abstract]

Received 22 May 2000; accepted 29 August 2000.