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J Virol. 2012 July; 86(13): 7241–7248.
PMCID: PMC3416320

Profound Amplification of Pathogenic Murine Polytropic Retrovirus Release from Coinfected Cells

Abstract

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98–F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.

INTRODUCTION

Infection of mice by exogenous ecotropic murine leukemia viruses (MuLVs) frequently results in the generation of recombinant viruses in which a portion of the envelope gene encoding the receptor-binding region has been replaced with the analogous envelope sequences derived from endogenous polytropic retroviruses (13, 6, 8, 11, 18, 20, 21, 27, 29, 39). The polytropic env gene encodes a protein (SU) that utilizes a cell surface receptor distinct from the ecotropic receptor, resulting in an altered infectious host range of the recombinants (4, 41, 46). Ecotropic MuLVs infect only cells of murine origin, whereas recombinant polytropic MuLVs infect murine cells as well as cells of numerous other species (16). Although some endogenous polytropic proviruses carry intact retroviral genes, replication-competent endogenous polytropic viruses have not been described (2, 39). The generation of recombinant polytropic MuLVs is thought to be instrumental in proliferative diseases resulting from ecotropic MuLV infection (7, 25, 35, 40, 4245). A principal mechanism of oncogenesis ultimately involves the integration of MuLVs near proto-oncogenes and may involve either the exogenous ecotropic virus or the recombinant polytropic MuLVs (5, 17, 32, 44, 45). It is also apparent that in vivo interactions of the viruses in the mixed infections generated by recombination strongly influence the pathogenicity of the retroviruses. In this regard, we have described in vivo interactions between ecotropic and recombinant polytropic MuLVs that may facilitate a stepwise process of leukemogenesis in mice infected with Moloney murine leukemia virus (M-MuLV) (29). More recently, we examined the interactions of ecotropic and polytropic MuLVs that were inoculated as virus mixtures into mice (22). Coinoculation of the ecotropic virus Friend murine leukemia virus (F-MuLV) with one polytropic virus (Fr54) greatly extended the survival times of mice compared to those of mice inoculated with F-MuLV alone, while coinoculation of F-MuLV with another polytropic virus (Fr98) induced a rapidly fatal neurological disease that was not observed in infections with either virus by itself (22). In both instances, we observed nearly complete pseudotyping of the polytropic virus RNA genome within ecotropic virions. Furthermore, the polytropic virus load was greatly elevated in coinoculated mice compared to that in mice inoculated with the polytropic virus alone. In contrast, the ecotropic virus load in coinoculated mice was unchanged. The processes leading to the extensive pseudotyping of polytropic MuLVs and the elevation of the polytropic virus load are not known, nor is it known if there is a relationship between pseudotyping and an elevated polytropic virus load.

In the present study, we examined coinfection of in vitro cell lines by ecotropic and polytropic MuLVs. Our in vitro observations are remarkably similar to what was observed in vivo and have enabled us to examine the interactions of retroviruses in mixed infections in a less complex setting. Our results indicate that the release of polytropic MuLV virion RNA from the cell is greatly facilitated by pseudotyping within ecotropic virions and strongly suggest that the recombinant polytropic MuLV Env proteins are less efficient participants in virion assembly processes than ecotropic MuLV Env proteins.

MATERIALS AND METHODS

Cells, viruses, and vectors.

NIH 3T3 cells were used for the propagation of viruses and for assays of MuLVs. All cells were maintained in tissue culture media supplemented with glutamine (2 mM), penicillin (100 units/ml), streptomycin (25 μg/ml), gentamicin (50 μg/ml), and amphotericin B (2 μg/ml). NIH 3T3 cells, the mouse microglial cell line 1094MG, and the neural stem cell line C17.2 (38) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated bovine calf serum. The 1094MG cell line was obtained from William Lynch, Iowa State University. Mus dunni cells (28) were maintained in RPMI medium containing 10% heat-inactivated fetal bovine serum. The ecotropic MuLV F-MuLV 57 (31), the amphotropic MuLV 4070A (24), and the polytropic MuLVs Fr98 (34) and Fr54 were obtained as virus stocks after transfection of NIH 3T3 cells with plasmids encoding each provirus and subsequent infection of Mus dunni cells. The ecotropic MuLVs M-MuLV 1387 and AKR 2A (13) were harvested from infected NIH 3T3 cells, and the polytropic MuLVs FrNx (1), 383-2T (21), M-RV 2A, and M-RV 7A (2) were harvested from infected Mus dunni cells. The AKV recombinant viruses AKR 13, MCF 247, and Akv-2-C34 (14) were originally obtained as virus stocks from Miles Cloyd and Janet Hartley (14). M73P-S is a polytropic virus isolated from AKR/J mice in our laboratory (23). Virus stocks of the AKV recombinants were harvested from chronically infected Mus dunni cells. The retroviral vector G1nβgSVNa (Gene Therapy Inc., Gaithersburg, MD) carries the Escherichia coli β-galactosidase (β-Gal) gene with the simian virus 40 (SV40) large T-antigen nuclear localization signal driven by the M-MuLV long terminal repeat. LAPSN (Clontech Laboratories, Inc., Palo Alto, CA) contains the human placental alkaline phosphatase (AP) gene driven by the M-MuLV long terminal repeat.

Infectivity, transduction assays, and MAbs.

Infectious MuLVs were quantified using monoclonal antibodies (MAb) specific for the SU proteins of the viruses in a focal immunofluorescence assay (FIA) (37). Briefly, NIH 3T3 cells were seeded and infected with serial dilutions of samples containing 8 μg/ml of Polybrene (Sigma-Aldrich). The cells were allowed to grow to confluence (5 days). The monolayers were incubated with MAbs (~0.1 ml per dish of medium harvested from hybridoma cell lines producing the MAb), rinsed, and then incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Ig) to bind to the MAbs on infected cells. Foci of infected cells were detected and quantified by fluorescence microscopy. The MAbs employed in the assays included MAb 538, which is specifically reactive with M-MuLV (29); MAb 48, which is specifically reactive with F-MuLV (10); and MAb 514 and MAb 516 (9), which react specifically with polytropic MuLVs. For transduction assays, NIH 3T3 cells were initially transfected with plasmids carrying the retroviral vector LAPSN or G1nβgSVNa and then cultured in the presence of G418 to select for transduced cells. The transduced cultures were then infected with the polytropic MuLV Fr98 to establish cell lines releasing progeny polytropic MuLVs as well as retroviral vectors pseudotyped within polytropic virions. The polytropic MuLVs released from the cultures were assessed using the FIA described above. Transduction by released vectors (LAPSN or G1nβgSVNa) encapsulated in MuLV virions was quantified as previously described, using assays for AP or β-Gal activity (26) in NIH 3T3 cells.

Pseudotyping of polytropic genomes within ecotropic or amphotropic virions was assessed by infectivity assays using uninfected target cells and cells which were chronically infected with ecotropic or amphotropic MuLVs. The chronically infected cells were completely refractory to infection by virions utilizing the same receptor, including virions containing pseudotyped polytropic genomes. The degree of pseudotyping was assessed by comparisons of infectivity for uninfected target cells susceptible to both pseudotyped and nonpseudotyped polytropic virions and infectivity for chronically infected cells susceptible only to nonpseudotyped polytropic virions. Similarly, pseudotyping of the LAPSN and G1nβgSVNa vectors was assessed by the AP or β-Gal assay on NIH 3T3 cells as well as on NIH 3T3 cells infected by the ecotropic MuLV, to assess the degree of pseudotyping of the vectors within ecotropic virions.

Cell surface staining and flow cytometry.

Cells infected with the polytropic virus Fr98 or infected with Fr98 and superinfected with F-MuLV were treated with phosphate-buffered saline (PBS) containing 0.002 M EDTA to detach the cells from the tissue culture plate. The cells were incubated with MAb 514, reactive with the SU protein of Fr98, followed by incubation with FITC-conjugated goat anti-mouse Ig. Similarly, cells infected with the ecotropic virus F-MuLV or infected with F-MuLV and superinfected with Fr98 were detached and incubated with MAb 48, reactive with the SU protein of F-MuLV, followed by incubation with FITC-conjugated goat anti-mouse Ig. Data were collected by an LSRII (BD Biosciences) flow cytometer and were analyzed using FlowJo (Tree Star).

Quantification of virion RNA.

Comparisons of the amounts of Fr98 virion RNA released from cells were accomplished using an RNase protection assay (RPA). Total RNA from a virus pellet equivalent to 1 ml of tissue culture supernatant was prepared using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was then analyzed for specific viral RNA by use of a Riboquant system (BD Biosciences). A probe specific to Fr98 envelope RNA was generated using the plasmid pc1579, which contained nucleotides 310 to 503 of the Fr98 envelope gene. The plasmid was linearized using EcoRI and transcribed with T7 polymerase, using Riboquant reaction mix and 32P-labeled UTP (NEN Life Sciences). The probe was quantified and then mixed with RNA samples. Samples containing the probe were heated to 90°C and then incubated at 56°C overnight. Following hybridization, samples were treated with RNase A, phenol-chloroform extracted, and ethanol precipitated following the Riboquant protocol (BD Biosciences). Samples were analyzed on a precast Tris-borate-EDTA (TBE)–urea gel (Bio-Rad). The 32P-labeled protected probe was quantified using a Storm phosphorimager (Amersham Biosciences, Piscataway, NJ) and ImageQuant software. Samples without viral RNA were used as a negative control to ensure RNase degradation of unbound labeled probe.

RESULTS

Amplification of polytropic MuLVs released from cell lines coinfected with ecotropic MuLVs.

In previous studies, we observed that inoculation of mice with mixtures of the ecotropic virus F-MuLV and the polytropic isolate Fr98 or Fr54 resulted in a profound elevation of the polytropic virus load (22, 33). To determine if this occurred in vitro, we compared infectious polytropic viruses released from cell lines infected with only the polytropic virus to polytropic viruses released from cells infected with both the polytropic virus and F-MuLV. Chronically infected NIH 3T3 cell lines were established that released stable levels of polytropic MuLVs. The cells were then superinfected with an ecotropic MuLV at a high multiplicity of infection to achieve complete ecotropic MuLV infection of the culture. This approach was taken to readily establish ecotropic viral interference, thereby minimizing reinfection of the cells by the polytropic MuLV because of ecotropic viral pseudotyping.

F-MuLV superinfection of NIH 3T3 cells chronically infected with either Fr54 or Fr98 resulted in a profound increase in the polytropic MuLV titer compared to that in chronically infected cells that were mock infected (Fig. 1A). This observation is consistent with our in vivo results indicating highly elevated polytropic MuLV replication in coinoculated mice (22, 33). Similar results were observed when M. dunni cells chronically infected with Fr98 were superinfected with F-MuLV.

Fig 1
Amplification of polytropic viruses but not ecotropic viruses released from coinfected cells. (A) NIH 3T3 cells chronically infected with the polytropic MuLV Fr54 or Fr98 were mock infected or superinfected with the ecotropic virus F-MuLV. The cells were ...

It was of interest to determine if the amplification of the polytropic MuLVs by coinfection with ecotropic viruses extended to virus combinations other than the ecotropic virus F-MuLV and its derivative polytropic recombinant viruses. We examined the effects of coinfections of various combinations of MuLVs. These included other ecotropic MuLVs coinfected with their derivative polytropic MuLVs, ecotropic MuLVs coinfected with polytropic MuLVs from a different ecotropic parent MuLV, and an amphotropic MuLV coinfected with polytropic MuLVs. The viruses used included both class I leukemogenic polytropic MuLVs and class II nonleukemic polytropic MuLVs derived from AKR mice (27, 32, 33).

The ecotropic MuLV component of in vitro mixed infections is not amplified.

Our previous in vivo studies of mixed infection revealed a profound elevation of the polytropic MuLV titer in coinfected mice compared to that in mice infected with only the polytropic MuLV. In contrast, the level of ecotropic MuLV in coinfected mice was the same as that in mice inoculated with only the ecotropic MuLV. To examine this phenomenon in vitro, cell lines chronically infected with the ecotropic virus F-MuLV were superinfected with the polytropic MuLV Fr98, and the ecotropic infectivity was determined. The level of ecotropic MuLV released from the coinfected cells was not significantly altered compared to that for mock-infected control cells (Fig. 1B).

Amplification of retroviral vectors released from coinfected cell lines.

To determine if the amplification of polytropic infectivity reflected a distinctive property of polytropic genomic RNA, we examined the effect of coinfection on the release of retroviral vectors from in vitro cell lines. Cell lines harboring retroviral vectors encoding either β-Gal (G1nβgSVNa) or AP (LAPSN) were infected with the polytropic MuLV Fr98 and passaged in culture several times to establish a chronic infection. Such cells release a mixture of virions, i.e., those which have packaged the vector genome and those which have packaged the polytropic Fr98 genome. The cells were then superinfected with F-MuLV, and the titers of the packaged vectors as well as the packaged Fr98 genome released from the superinfected cell lines were compared to titers released from cells infected with the polytropic virus alone. All titers were assessed using uninfected NIH 3T3 cells and the AP assay, the β-Gal assay, or an FIA using MAb 514, which is reactive with the polytropic SU protein. With both vectors, a greatly elevated transduction titer was observed for viruses released from the coinfected cells, and it closely paralleled the increase in polytropic MuLV titers measured by FIA (Fig. 2). These results indicate that amplification of infectivity of the polytropic MuLV in mixed infections is not due to an inherent property of the polytropic MuLV RNA.

Fig 2
Release of polytropic MuLV and retroviral vectors after superinfection with F-MuLV. NIH 3T3 cells harboring the retroviral vector LAPSN or G1nβgSVNa and chronically infected with the polytropic MuLV Fr98 were mock infected or superinfected with ...

Polytropic MuLV genomes released from coinfected cells are extensively pseudotyped within ecotropic virions.

Polytropic viral genomes released from coinfected mice are extensively pseudotyped by ecotropic MuLVs (22). This is true with coinoculated mice as well as with mice which have generated polytropic viruses after infection with an ecotropic MuLV (29, 36). To determine if the polytropic MuLVs released from coinfected in vitro cell lines are also pseudotyped within ecotropic virions, the infectious titers of polytropic MuLVs were assessed on uninfected NIH 3T3 cells as well as NIH 3T3 cells that were chronically infected with the ecotropic MuLV. Polytropic MuLV genomes packaged in polytropic virions or pseudotyped within ecotropic virions can infect uninfected target cells because the receptors recognized by the SU proteins of the polytropic or ecotropic virions are not blocked. However, target cells chronically infected with the ecotropic MuLV are refractory to infection by all ecotropic virions, including those containing pseudotyped polytropic RNA genomes. Thus, extensively pseudotyped polytropic viruses exhibit a greatly reduced titer on ecotropic MuLV-infected cells, in which the ecotropic MuLV receptor is blocked, in contrast to the case with uninfected target cells. The polytropic MuLVs released from all of the cultures coinfected with ecotropic and polytropic MuLVs exhibited extensive pseudotyping of the polytropic MuLV (Table 1). Amplification of the retroviral vectors described earlier (Fig. 2) was also associated with extensive pseudotyping of the vectors within ecotropic virions (data not shown).

Table 1
Amplification and pseudotyping of polytropic MuLVs in mixed infections

Amplification and pseudotyping of the polytropic virus Fr98 are observed in cells of CNS origin.

All of the in vitro experiments described above employed fibroblastic cell lines. It was of interest to determine if the results could be extended to other cell types. In previous studies, we found that coinfection of Fr98 and F-MuLV resulted in a severe neurological disease that was not seen when either virus was inoculated alone (22). The disease, which culminated as early as 10 days after infection, was associated with a massive infection of the central nervous system (CNS) by the polytropic Fr98 virus that was not observed in mice infected with Fr98 alone. Thus, it seemed appropriate to determine if the amplification and pseudotyping observed upon coinfection of fibroblasts occurred in cells of CNS origin. A microglial cell line (1094MG) and a neural stem cell line (C17.2) were infected with Fr98, and the cultures were passaged several times to establish chronic infections. The cells were then superinfected with F-MuLV or mock infected, and the progeny viruses released from the cultures were assessed for polytropic virus titers as well as for pseudotyping of Fr98 within ecotropic virions. With both CNS cell lines, we observed amplification of the polytropic virus that was quite similar to our observations with fibroblasts (Fig. 3). The level of Fr98 pseudotyped with ecotropic virions was >95% for the C17.2 neural stem cell line and >97% for the 1094MG microglial cells. Similar results were obtained using Fr54, which does not induce a rapid neurological disease (22, 33).

Fig 3
Polytropic viruses released from CNS cell lines chronically infected with the polytropic MuLV Fr98 after superinfection with the ecotropic virus F-MuLV. The microglial cell line 1094MG and the neural stem cell line C17.2 were chronically infected with ...

Cell surface expression of the polytropic surface envelope protein (SU) is depressed on coinfected cells.

The large increase in the infectious titers of polytropic viruses appears to be the result of pseudotyping, but coinfected cells continue to release nonpseudotyped polytropic virus at levels only moderately lower than those in cells infected with the polytropic MuLV alone. This was evident from assessments of the levels of polytropic MuLV infection on target ecotropic MuLV-infected cells (Fig. 4). These target cells are refractory to infection by ecotropic virions but permissive to infection by polytropic virions and thus provide a measure of the level of virions utilizing the polytropic SU protein. The decrease in polytropic virion release in coinfected cells correlated with a decreased level of polytropic SU on the surfaces of cells, as assessed by flow cytometry analyses using a monoclonal antibody specifically reactive to the protein. These observations are consistent with the possibility that the level of cell surface SU influenced the level of nonpseudotyped polytropic virions released from the cells (Fig. 5A). The level of cell surface F-MuLV ecotropic SU protein was unchanged on coinfected cells (Fig. 5B).

Fig 4
Decrease in nonpseudotyped polytropic virions released after superinfection with F-MuLV. NIH 3T3 cells chronically infected with the polytropic MuLV Fr98 were mock infected or superinfected with the ecotropic virus F-MuLV. The cells were grown to confluence, ...
Fig 5
Cell surface expression of SU proteins after superinfection of cells chronically infected with F-MuLV or Fr98. (A) NIH 3T3 cells infected with the polytropic virus Fr98 were subsequently coinfected with the ecotropic virus F-MuLV (solid line) or mock ...

Increased efficiency of packaging of the Fr98 genome contributes to the amplification of Fr98 after coinfection with F-MuLV or amphotropic MuLV.

The marked increase in polytropic MuLV titers attributed to pseudotyped polytropic viruses could proceed by at least two mechanisms. If the ecotropic virion were exceedingly more infectious than the polytropic virion, pseudotyping of the polytropic genomic RNA within the ecotropic virion would result in a greatly increased polytropic MuLV titer. A second mechanism, not contingent upon differences in infectivity, could be an increased efficiency of virion assembly and RNA packaging by the ecotropic MuLV compared to the polytropic MuLV. In contrast to an increase in specific infectivity, an increase in the efficiency of polytropic RNA packaging would be reflected in the level of virion polytropic RNA released from the cell. To examine these possibilities, we quantified the polytropic RNA released from cells coinfected with Fr98 and F-MuLV, cells coinfected with Fr98 and the amphotropic virus 4070A, and cells infected with Fr98 alone. In addition, we assessed the polytropic virus titers and the degree of pseudotyping of the polytropic genome within ecotropic or amphotropic virions. A close correlation was observed between the level of polytropic infectivity and the level of virion polytropic RNA released from the cells (Fig. 6). Coinfection with either F-MuLV or 4070A resulted in >99% pseudotyping of the Fr98 genome.

Fig 6
Levels of polytropic Fr98 infectivity and virion RNA released from Fr98-infected cultures superinfected with the ecotropic virus F-MuLV or the amphotropic MuLV 4070A. NIH 3T3 cells chronically infected with the polytropic MuLV Fr98 were mock infected ...

DISCUSSION

Our previous studies of in vivo mixed retrovirus infections revealed remarkably altered pathology compared to that after infection with individual viruses of the mixture (19, 22). In those studies, the polytropic virus load in the coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, the polytropic MuLV was nearly completely pseudotyped within ecotropic virions in coinoculated mice. It was possible that these observations reflected events restricted to in vivo infection, e.g., spread of the polytropic MuLV to different cell types facilitated by pseudotyping within ecotropic virions, ecotropic MuLV-induced expansion of target cells for the polytropic virus, or infection of cells that preferentially assemble ecotropic rather than polytropic envelope proteins into virions. Alternatively, some of these observations may reflect more basic features of coinfection by ecotropic and polytropic viruses, independent of the complex in vivo environment.

Our in vitro studies revealed a striking elevation of the polytropic MuLV infectivity released from coinfected cells compared to that released from cells infected with the polytropic MuLV alone. In all experiments, amplification was restricted to the polytropic MuLV component of the mixture, similar to our earlier in vivo results (22). It is apparent that this is a general phenomenon, as amplification of the polytropic MuLV was observed with many different polytropic-ecotropic MuLV pairs as well as with polytropic-amphotropic MuLV mixtures. In all instances, the amplification of the polytropic titer was accompanied by extensive pseudotyping of the polytropic genome within ecotropic or amphotropic virions. Thus, all or nearly all of the increase in polytropic MuLV infectivity observed was likely the result of pseudotyped polytropic MuLV genomes. Furthermore, the increased infectivity closely paralleled the level of polytropic virion RNA released from cells, indicating an increase in infectious particles rather than a large difference in specific infectivity between polytropic and ecotropic virions.

The explanation for the predominant packaging of the polytropic genome into ecotropic particles is unclear. Although polytropic and ecotropic SU proteins are present on coinfected cells, most released virions exhibit an ecotropic host range. It is possible that virions are mosaic with respect to the SU proteins and that the infectivity of virions depends on the density of the receptor-binding SU proteins on the virion surface. The ecotropic SU protein, if present in a large excess over the polytropic SU protein, might simply dilute out the polytropic SU protein below a threshold density required for infection. If this were the case, one would predict that the release of nonpseudotyped polytropic viruses would be severely depressed. Our results do not support this hypothesis. We found that ecotropic pseudotyping of the polytropic genome did not severely supplant the release of polytropic virions, as evidenced by the continued release of virions utilizing the polytropic SU protein in coinfected cells (Fig. 4). Rather, pseudotyping of the polytropic genome within ecotropic virions exerted an additive effect, augmenting the total polytropic infectious titer (pseudotyped and nonpseudotyped) released from the coinfected cells.

A more likely explanation for the predominance of ecotropic virions in mixed infections is that the polytropic SU protein participates less efficiently in the virion assembly process than the ecotropic SU protein. Polytropic viruses are chimeric recombinant viruses comprised of genes from different retroviral genomes which have diverged along different evolutionary pathways. Indeed, many recombinant polytropic MuLVs encode a chimeric SU protein consisting of sequences derived from both the endogenous polytropic and the exogenous ecotropic parent. A chimeric protein may function less efficiently than either of the parental proteins. In this regard, functional impairment has been observed in chimeric viruses constructed between very closely related ecotropic MuLVs (30).

The decrease in cell surface polytropic SU protein in coinfected cells may be pertinent to the moderately reduced levels of nonpseudotyped polytropic viruses released from the cell. The decreased cell surface polytropic SU protein on coinfected cells could reflect competition for processing through the Golgi apparatus or competition for deposition on available plasma membrane sites. It is also possible that the ecotropic virus interferes at other stages in the polytropic MuLV replication cycle, such as RNA splicing to generate env mRNA or transport of RNA to the cytoplasm. Investigations of these aspects of interactions between viruses in mixed infections should be facilitated greatly in an in vitro system that mirrors in vivo mixed infections.

We found that the levels of polytropic virion RNA released from cells infected with Fr98 or superinfected with either F-MuLV or an amphotropic MuLV correlated closely with the infectious polytropic titers released from the cells (Fig. 6). Furthermore, the polytropic genome was extensively pseudotyped in coinfections with either ecotropic or amphotropic MuLVs. It is possible that superinfection by either virus results in an elevated level of polytropic transcripts available for packaging. However, we observed nearly identical amplification of the polytropic MuLV titers and the titers of two different retroviral vectors after superinfection with F-MuLV (Fig. 2). It seems unlikely that transcriptional activation of different integrants would account for precisely the same elevation of transduction or infection. Rather, these results further support the premise that the increased efficiency in the assembly process of the ecotropic virus compared to the polytropic virus is responsible for the elevated polytropic virion RNA level.

Although differences in assembly efficiency contribute to the increase in infectious virus observed in these studies, it is likely that the specific infectivity of different virions also contributes. In this regard, recombinant polytropic MuLVs differ greatly in their ability to infect mouse cells (12, 20, 23). Ecotropic pseudotyping of a poorly infectious polytropic MuLV would be expected to increase its relative infectivity to a greater extent than pseudotyping of a polytropic virus that readily infects mouse cells.

In this study, we examined coinfection of in vitro cell lines by ecotropic and polytropic MuLVs. Our in vitro observations are remarkably similar to what we observed previously in vivo. Furthermore, these observations extend to different in vitro cell lines, including cells of CNS origin which are targets for the polytropic MuLV Fr98. Our studies strongly suggest that the effects of mixed infections on replication and pathology in the host reflect fundamental features of the interactions among retroviruses in mixed infections. Retroviral mixed infections are common in nature, and it is clear that interactions among retroviruses can have profound pathological consequences. In this regard, our results may be pertinent to early studies of polytropic viruses from AKR mice as well as from certain other mouse strains (14, 15). In those studies, which identified both pathogenic and nonpathogenic viruses, pathogenic recombinant viruses were identified by examining the ability to accelerate disease in mice which harbor and express high levels of endogenous ecotropic MuLVs. The pathogenicity of the viruses was not apparent in mouse strains that did not express ecotropic MuLVs, suggesting that mixed infection was a prerequisite for pathogenicity. Considering that pseudotyping and amplification of polytropic MuLVs are observed as general occurrences with a variety of virus combinations and with different cell types, it seems quite likely that these interactions underpin many pathological consequences of mixed retrovirus infections. Further studies using in vitro cell lines that mimic in vivo mixed infections should facilitate a more thorough understanding of these interactions.

ACKNOWLEDGMENTS

We thank K. Hasenkrug, S. Priola, G. Baron, and B. Caughey for helpful discussions and Ron Messer for flow cytometric analyses.

This research was supported by the intramural research program of the NIAID, NIH.

Footnotes

Published ahead of print 18 April 2012

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