Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells

Lars Aagaard1, Jacob Giehm Mikkelsena,1, Søren Warming1, Mogens Duch1 and Finn Skou Pedersen1,2

Department of Molecular and Structural Biology1, Department of Medical Microbiology and Immunology2, C. F. Moellers Allé, Bldg 130, DK-8000 Aarhus, University of Aarhus, Denmark

Author for correspondence: Finn Skou Pedersen. Fax +45 86196500. e-mail fsp{at}mbio.aau.dk


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To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1b cells with respect to infection though the ecotropic receptor pathway.


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As part of the retrovirus life cycle a DNA copy of the RNA genome is inserted into the host genome. During the course of evolution multiple retrovirus infections into the germline of vertebrates such as mammals and birds have accumulated vast amounts of endogenous retroviruses (ERV). In the human genome ~8% is composed of ERVs or related sequences (Genome International Sequencing Consortium, 2001 ). Examples from mice have shown that the host can adopt ERV proteins as a defence against retrovirus infections. One prominent example is the Fv1 locus, discovered more than three decades ago (Lilly, 1970 ). Fv1 encodes a capsid (CA)-like protein (Best et al., 1996 ) that restricts murine leukaemia virus (MLV) infection at a post-entry preintegration level (Pryciak & Varmus, 1992 ). This CA-like protein most likely derived from the ERV-L family (Bénit et al., 1997 ) in early Mus speciation (Qi et al., 1998 ), which makes it distantly related to its MLV target.

Fv1 is found in two main allelic versions, Fv1n and Fv1b, present in NIH Swiss and BALB/c mice respectively. The Fv1n allele restricts replication of B-tropic MLV while allowing replication of N-tropic viruses and vice versa. NB-tropic viruses infect mice having either allele. The amino acid at CA position 110 determines if a virus is N- (Arg) or B-tropic (Glu) (Kozak & Chakraborti, 1996 ). It has been shown that the Fv1 alleles are codominant in such a way that Fv1n/b mice restrict both N- and B-tropic viruses. Likewise, mixed virus particles harbouring both N- and B-tropic determinants will be sensitive to both Fv1 alleles. The restriction of infection in non-permissive cells is not absolute, but leads to a 50–1000 decrease in virus titre. It is believed that Fv1 restriction can be relieved, or abrogated, if the same cell is hit by two or more virus particles (Duran-Troise et al., 1977 ). However, the mechanism underlying Fv1 restriction remains unclear.

Recently, Towers et al. (2000) published the intriguing observation that a wide range of non-murine cells restricts N-tropic viruses in an Fv1-like manner. This study used single-round transfer of vesicular stomatitis virus G protein (or amphotropic) pseudotyped retrovirus vectors to infect mammalian cells using both N- and B-tropic packaging constructs. It was shown that the restriction was dependent on CA position 110 and that mixed virus particles were sensitized. The gene responsible for the restriction was dubbed REF1 (restriction factor 1). Using a different approach, we could detect this restriction in the context of full-length ecotropic replication-competent MLV infecting mCAT-1-expressing human cells as compared to infections with pseudotyped vector preparations. We also included NB-tropic (as well as N- and B-tropic) determinants in our study. Part of our work took advantage of an EGFP-expressing virus that allowed us to detect the infection of a virus harbouring a full-length genome. We here present data that support and strengthen the existence of Fv1-like restriction in human cells.

In an initial series of experiments we observed that N-tropic Akv MLV failed to replicate in a human cell line (U2OS; Ponten & Saksela, 1967 ) modified to stably express the ecotropic mCAT-1 receptor, whereas the NB-tropic Moloney MLV replicated efficiently. We tested this more rigorously by using SL3-3 and SL3-3NB MLV. The parental SL3-3 virus is N-tropic whereas the derived SL3-3NB was turned NB-tropic by insertion of Moloney sequence at the N-terminal region of CA (position 1–135) leading to five amino acid substitutions conferring the NB tropism (Thomas et al., 1993 ). Target cells were infected with equal amounts of these four viruses (normalized by reverse transcriptase assay) in the presence of 6 µg polybrene per ml and passaged under standard conditions for 18 days in order to allow virus spread. Virus production was quantified from supernatant by reverse transcriptase assay (Lovmand et al., 1998 ) and normalized to the total number of cells (Fig. 1). Similar results were obtained when using 10-fold more or less virus (data not shown). Direct transfection of infectious N-tropic Akv DNA into the mCAT-1-expressing U2OS cells likewise did not lead to spread of virus even after prolonged cell culturing (>50 days, data not shown). To rule out contamination of cell stocks we amplified the GADPH gene by PCR from U2OS genomic DNA and verified by sequencing the gene of human origin. Sequencing of RNA (cDNA) isolated from virus particles likewise verified that no virus contamination nor spontaneous mutations had occurred within the CA region during the experiment.



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Fig. 1. Replication efficiency of N-tropic MLV Akv and SL3-3 and NB-tropic Moloney and SL3-3NB virus (Thomas et al., 1993 ) on murine (NIH/3T3, BALB/3T3) and human mCAT-1-expressing cells (U2OS). Hatched and black bars represent N- and NB-tropic viruses respectively.

 
As shown in Fig. 1, infection of U2OS cells with either N-tropic Akv or SL3-3 MLV did not cause any detectable virus production whereas infection with NB-tropic Moloney and SL3-3NB did. Note that the infections with Akv and SL3-3 were at levels sufficient to overcome the Fv1 restriction in BALB cells. However, a 10-fold dilution did cause reduced virus release from BALB cells infected with restricted (N-tropic) viruses when sampled at early time-points (data not shown). The efficient replication of SL3-3NB versus SL3-3 in human cells testifies that NB-tropic viruses, like B-tropic viruses (Towers et al., 2000 ), escape the REF1 restriction imposed on N-tropic viruses.

To further address the escape requirements of viruses using the ecotropic entry pathway in human cells, we took advantage of an EGFP-expressing virus (AkvU3-EGFP) that allows direct titre measurement (Jespersen et al., 1999 ; Bachrach et al.,2002 ). This N-tropic virus was modified in the CA region to alter the tropism. The B-tropic variant, AkvBU3-EGFP, was changed at genome position 1608–1609 from AG to GA resulting in an Arg to Glu substitution at CA position 110. This point mutation was introduced into a PCR fragment which was subsequently cloned into the flanking SacII and DraIII sites (Fig. 2A). The NB-tropic variant, AkvNBU3-EGFP, had a Moloney fragment inserted at position 1281–1685, resulting in the following amino acid changes in the N-terminal region of CA: position 4, Leu to Ala; position 46, Thr to Ile; position 82, Asn to Asp; position 110, Arg to Ala; and position 117, Leu to His. The substitution was generated by extending a Moloney PCR fragment encompassing CA position 1–135 (forward primer harbouring an Akv-derived linker from SacII to the p12/CA border) with an Akv PCR fragment (with the DraIII site) overlapping at position 1675–1695. The PCR product was cloned into the SacII and DraIII sites of Akv-U3EGFP.



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Fig. 2. (A) Schematic diagram of EGFP-expressing viruses and the titre assay used in this study. Arrows indicate introduced mutations and appropriate cloning sites. (B) Titres of EGFP-expressing B- or NB-tropic viruses relative to N-tropic viruses on different human cells and murine control cells. B/N (black bars) or NB/N (hatched bars) values were obtained by dividing the titre of B-tropic virus (AkvBU3-EGFP) or NB-tropic virus (AkvNBU3-EGFP) with titres of N-tropic virus (AkvU3-EGFP) respectively after normalization to SC-1 titres (as shown in Table 1). Standard errors are indicated (n=3).

 
We tested the titre of these viruses (AkvU3-EGFP, AkvBU3-EGFP and AkvNBU3-EGFP) on a series of murine (NIH/3T3, BALB/3T3 and SC-1; Hartley & Rowe, 1975 ) and mCAT-1-expressing human cells [AMA (Celis et al., 1987 ), 293, HeLa and U2OS]. Briefly, infectious stocks of EGFP-expressing viruses were used to infect target cells (seeded at 5–10x103 per cm2 in 12-well plates) in the presence of 6 µg polybrene per ml (Fig. 2A). The following day anti-envelope antibody (83A25; Sitbon et al., 1985 ) was added to prevent secondary spread. Three days post-infection the number of EGFP-expressing foci was determined using a fluorescence microscope (Olympus, HQ:F801 filter) and the resulting titre was normalized to Fv1-null SC-1 cells. Table 1 shows that both B- and NB-tropic versions readily infect the human U2OS cells as compared to the low titres with N-tropic AkvU3-EGFP. Similar results were obtained using the remaining human cell lines and the results are summarized in Fig. 2(B). Notably, the level of restriction varies more than 100-fold among the human cells. This may be due to variation in expression of the responsible gene(s) and/or allelic differences among the human cells. Although the REF1 restriction seems difficult to overcome in the U2OS cells, the remaining human cell lines tested showed no absolute restriction and thus behaved like Fv1b cells. The single point mutations made between AkvU3-EGFP and AkvBU3-EGFP confirm the dependence of REF1 (and Fv1) restriction on CA position 110 (Towers et al., 2000 ) while the escape with Moloney-derived NB determinants further supports the resemblance to Fv1.


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Table 1. Titres of EGFP-expressing viruses on murine and human cells after normalization to Fv1-null cells

 
The mechanism of Fv1 restriction is still poorly understood. It has been argued that it involves direct interactions between CA structures in the incoming virus and the Fv1 protein (Goff, 1996 ). The observation that neither Fv1n cells pretreated with NB-tropic virions nor Fv1n cells expressing NB-tropic Gag proteins could affect the action of Fv1 on N- and B-tropic virus challenge (L. Aagaard, J. G. Mikkelsen, M. Duch & F. S.Pedersen, unpublished results), is compatible with a hypothesis of direct binding. However, other possibilities such as competitive binding for a cellular target cannot be excluded. REF1 has not been described in genetic terms and only by analogy to Fv1 can it be speculated that REF1 possibly encodes a CA-like molecule with the same intriguing properties as Fv1. If so, this might be an example of human co-option of ERV debris as it could provide a selective advantage against retroviral pathogens.

We have thus confirmed the Gag-defined post-entry block to N-tropic virus infection in human cells as observed by Towers et al. (2000) , and further extended the analogy between REF1 and Fv1 in two important ways: (i) by finding that NB-tropic determinants also escape restriction and (ii) by using full-length ecotropic MLV instead of pseudotyped vectors (Towers et al., 2000 ).


   Acknowledgments
 
This work was supported by the Danish Cancer Society, the Danish Natural Sciences and Medical Research Councils and the Karen Elise Jensen Foundation. We thank J. Lenz for kindly providing us with SL3-3NB.


   Footnotes
 
a Present address : Department of Genetics, School of Medicine, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.


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Received 29 August 2001; accepted 25 October 2001.