Neural Stem Cells as Engraftable Packaging Lines Can Mediate Gene Delivery to Microglia: Evidence from Studying Retroviral env-Related Neurodegeneration

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896-8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.     

Unique N-Linked Glycosylation of CasBrE Env Influences Its Stability,Processing, and Viral Infectivity but Not Its Neurotoxicity

The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 10² to 10⁵ FFU/ml); and wild-type-like (WTL) (titer > 10⁵ FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

G541R within the 4070A TM protein regulates fusion in murine leukemia viruses

The mutation G541R within the ectodomain of TM was isolated in three independent chimeric enveloped murine leukemia virus (MuLV) viral populations originally impaired in viral passage and in wild-type 4070A. Isolation of G541R in multiple populations suggested it played a critical role in viral envelope function. Using a viral vector system, the observed effects of the G541R mutation within MuLV envelope proteins were pleiotropic and included effects on the regulation of SU-TM interactions and membrane fusion. G541R suppresses enhanced cell-cell fusion events attributable to the absence of the R-peptide yet does not adversely affect virus titers. The ability to suppress cell-cell fusion is dependent on the presence of the C terminus of the amphotropic 4070A SU protein. Within the wild-type 4070A envelope background, the mutation results in a decreased level of Env at the cell surface that is mirrored in the virion. The TM mutation alters recognition of the SU C terminus by a monoclonal antibody, suggestive of an altered conformation. The presence of G541R allowed the virus to achieve a balance between cytopathogenicity and replication and restored productive viral entry.     

Late virus replication events in microglia are required for neurovirulent retrovirus-induced spongiform neurodegeneration: evidence from neural progenitor-derived chimeric mouse brains.

CasBrE is a neurovirulent murine retrovirus which induces a spongiform myeloencephalopathy in susceptible mice. Genetic mapping studies have indicated that sequences responsible for neurovirulence reside within the env gene. To address the question of direct envelope protein neuroxicity in the central nervous system (CNS), we have generated chimeric mice expressing the CasBrE envelope protein in cells of neuroectodermal origin. Specifically, the multipotent neural progenitor cell line C17.2 was engineered to express the CasBrE env gene as either gp70/p15E (CasE) or gp70 alone (CasES). CasE expression in these cells resulted in complete (>10(5)) interference of superinfection with Friend murine leukemia virus clone FB29, whereas CasES expression resulted in a 1.8-log-unit decrease in FB29 titer. Introduction of these envelope-expressing C17.2 cells into the brains of highly susceptible IRW mice resulted in significant engraftment as integral cytoarchitecturally correct components of the CNS. Despite high-level envelope protein expression from the engrafted cells, no evidence of spongiform neurodegeneration was observed. To examine whether early virus replication events were necessary for pathogenesis, C17.2 cells expressing whole virus were transplanted into mice in which virus replication in the host was specifically restricted by Fv-1 to preintegration events. Again, significant C17.2 cell engraftment and infectious virus expression failed to precipitate spongiform lesions. In contrast, transplantation of virus-expressing C17.2 progenitor cells in the absence of the Fv-1 restriction resulted in extensive spongiform neurodegeneration by 2 weeks postengraftment. Cytological examination indicated that infection had spread beyond the engrafted cells, and in particular to host microglia. Spongiform neuropathology in these animals was directly correlated with CasBrE env expression in microglia rather than expression from neural progenitor cells. These results suggest that the envelope protein of CasBrE is not itself neurotoxic but that virus infectious events beyond binding and fusion in microglia are necessary for the induction of CNS disease.     

Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.     

G100R mutation within 4070A murine leukemia virus Env increases virus receptor binding,kinetics of entry,and viral transduction efficiency

Passage of 4070A murine leukemia virus (MuLV) in D17 cells resulted in a G-to-R change at position 100 within the VRA of the envelope protein (Env). Compared with 4070A MuLV, virus with the G100R Env displayed enhanced binding on target cells, internalized the virus more rapidly, and increased the overall viral titer in multiple cell types. This provides a direct correlation between binding strength and efficiency of viral entry. Deletion of a His residue at the SU N terminus eliminated the transduction efficiency by the G100R virus, suggesting that the G100R virus maintains the regulatory characteristics of 4070A viral entry. The improved transduction efficiency of G100R Env would be an asset for gene delivery systems.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag-pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

The critical N-linked glycan of murine leukemia virus envelope protein promotes both folding of the C-terminal domains of the precursor polyprotein and stability of the postcleavage envelope complex.

The infectivity of Friend ecotropic murine leukemia virus was previously shown to be highly sensitive to modification in its envelope protein (Env) at only one of the eight signals for N-linked glycan attachment, the fourth from the N terminus (gs4). In the present study, a set of six single-amino-acid substitutions in or near gs4 was used to determine the function of this region of Env and the role played by the glycan itself. One mutant that lacked the gs4 glycan was fully infectious, while one that retained this glycan was completely noninfectious, indicating that the gs4 glycan per se is not required for Env function. Infectivity correlated with the level of mature Env complex incorporated into virus particles, which was determined by the severity of defects in transport of the envelope precursor protein (gPrEnv) from the endoplasmic reticulum into the Golgi apparatus, in cleavage of gPrEnv into the two envelope subunits (the surface protein [SU] and the transmembrane protein [TM]), and in the association of SU with cellular membranes. All of the mutants induced the wild-type level of superinfection interference, indicating that the gs4 region mutations did not interfere with proper folding of the N-terminal domain of SU. These results suggest that the gs4 region mediates folding of the C-terminal domains of gPrEnv and stability of the interaction between SU and TM. Although the gs4 glycan was not essential for infectivity, processing of all mutant Envs lacking this glycan was significantly impaired, suggesting that efficient folding of gPrEnv requires a glycan at this position. The conservation of a glycosylation site homologous to gs4 across a broad range of retroviruses suggests that this sequence may play a similar role in many retroviral Envs.     

Role of the mutation Q252R in activating membrane fusion in the murine leukemia virus surface envelope protein

Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.     

Gene transfer into mammalian cells by a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus.

We have constructed and characterized a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus (MLV). The chimeric retroviral genome was created by replacing the env coding region in the replication-competent retroviral vector RCASBP(A) with the env region from an amphotropic MLV. The recombinant vector RCASBP-M(4070A) forms particles containing MLV Env glycoproteins. The vector replicates efficiently in chicken embryo fibroblasts and is able to transfer genes into mammalian cells. Vector stocks with titers exceeding 10(6) CFU/ml on mammalian cells can be easily prepared by passaging transfected chicken embryo fibroblasts. Since the vector is inherently defective in mammalian cells, it appears to have the safety features required for gene therapy.     

Mutations of a residue within the polyproline-rich region of Env alter the replication rate and level of cytopathic effects in chimeric avian retroviral vectors

Previous attempts to extend the host range of the avian sarcoma/leukosis virus (ASLV)-based RCASBP vectors produced two viral vectors, RCASBP M2C (4070A) and RCASBP M2C (797-8), which replicate using the amphotropic murine leukemia virus 4070A Env protein (2). Both viruses were adapted to replicate efficiently in the avian cell line DF-1, but RCASBP M2C (4070A) caused extensive cytopathic effects (CPE) in DF-1 cells whereas RCASBP M2C (797-8) induced low levels of CPE. The two viruses differed only at amino acid 242 of the polyproline-rich region in the surface (SU) subunit of the Env protein. In RCASBP M2C (4070A), an isoleucine replaced the wild-type proline residue, whereas a threonine residue was found in RCASBP M2C (797-8). In the present study, we show that other amino acid substitutions at position 242 strongly influence the CPE and replication rate of the chimeric viruses. There was a correlation between the amount of unintegrated linear retroviral DNA present in infected DF-1 cells and the level of CPE. This suggests that there may be a role for superinfection in the CPE. The treatment of RCASBP M2C (4070A)-infected cells with dantrolene, which inhibits the release of calcium from the endoplasmic reticulum (ER), reduced the amount of CPE seen during infection with the highly cytotoxic virus. Dantrolene treatment did not appear to affect virus production, suggesting that Ca2+ release from the ER had a role in the CPE caused by these viruses.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

Stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system.     

TR1.3 viral pathogenesis and syncytium formation are linked to Env-Gag cooperation

Infection with murine leukemia virus (MLV) TR1.3 or the related molecular construct W102G causes severe neuropathology in vivo. Infection is causally linked to the development of extensive syncytia in brain capillary endothelial cells (BCEC). These viruses also induce cell fusion of murine cell lines, such as SC-1 and NIH 3T3, which are otherwise resistant to MLV-induced syncytium formation. Although the virulence of these viruses maps within the env gene, the mechanism of fusion enhancement is not fully determined. To this end, we examined the capacity of the syncytium-inducing (SI) TR1.3 and W102G MLVs to overcome the fusion inhibitory activity inherent in the full-length Env cytoplasmic tail. These studies showed that the TR1.3 and W102G Envs did not induce premature cleavage of p2E, nor did they override p2E fusion inhibition. Indeed, in the presence of mutations that disrupt p2E function, the TR1.3 and W102G Envs significantly increased the extent of cell fusion compared to that with the non-syncytium-inducing MLV FB29. Surprisingly, we also observed that TR1.3 and W102G Envs failed to elicit syncytium formation in these in vitro assays. Coexpression of gag-pol with env restored syncytium formation, and accordingly, mutations within gag-pol were used to examine the minimal functional requirements for the SI phenotype. The results indicate that both gag-dependent particle budding and cleavage of p2E are required to activate the SI phenotype of TR1.3 and W102G viruses. Collectively, these data suggest that the TR1.3 and W102G viruses induce cell fusion by the fusion-from-without pathway.     

Green fluorescent protein-tagged retroviral envelope protein for analysis of virus-cell interactions

Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.     

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.