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
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Abstract |
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Introduction |
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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 Freunds 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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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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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.
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Acknowledgments |
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References |
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Received 22 May 2000;
accepted 29 August 2000.