Capsid-dependent and -independent postentry restriction of primate lentivirus tropism in rodent cells

Retrovirus tropism can be restricted by cellular factors such as Fv1, Ref1, and Lv1 that inhibit infection by targeting the incoming viral capsid. Here, we show that rodent cells exhibit differential sensitivity to infection by vesicular stomatitis virus G-pseudotyped lentiviruses and that differences between human immunodeficiency virus type 1 and simian immunodeficiency virus (SIVmac) infectivity are sometimes, but not always, governed by determinants in capsid-p2. In at least one case, resistance to SIVmac infection could be eliminated by saturation of target cells with noninfectious SIVmac particles. However, cross-saturation experiments and analysis of Fv1-null cells engineered to express natural or artificial Fv1 proteins revealed that lentivirus restriction in mouse cells is independent of Fv1. Overall, these findings indicate that novel restriction factors in rodents can modulate sensitivity to specific primate lentiviruses.     

Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA "coat" can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.     

Capsid processing requirements for abrogation of Fv1 and Ref1 restriction

Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.     

Identification of the regions of Fv1 necessary for murine leukemia virus restriction

The Fv1 gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the Fv1 gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.     

Restriction factors: a defense against retroviral infection

Susceptibility to retroviral infection is determined, in part, by host genes with antiviral activity. The Fv1 gene, which inhibits murine leukemia virus infection in mice, encodes one such resistance factor, and was long thought to be unique in that it restricts post-entry, pre-integration steps of retroviral replication. However, recent findings suggest the existence of similar restriction factors in primates, including humans. These factors, termed Lv1 and Ref1, can inhibit a range of retroviruses, including human immunodeficiency virus type 1 and its relatives. Fv1, Lv1 and Ref1 target capsid determinants to block infection but can be saturated by incoming virions. Primate- and murine-retrovirus restriction factors have diverse and overlapping specificities, and some variants of Lv1, as well as Ref1, apparently recognize and inhibit infection by widely divergent retroviruses.     

A phenotypic host range alteration determines RD114 virus restriction in feline embryonic cells.

We have characterized the restriction mechanism for RD114 virus replication in embryonic feline cells (FeF). By comparing growth properties of the virus in FeF cells with its behavior in a fetal feline glial cell line (G355) permissive for RD114, we showed that both cell lines were readily infectible by virus grown in permissive cells and that no significant differences in viral integration or viral RNA expression could be detected. However, analysis of viral protein expression revealed differences in viral env gene processing in the two cell types. Envelope precursor pR85 was produced, but the expected processed gp70 product was detectable only in permissive (G355) cells. An envelope product of 85 kDa was packaged into virions produced by FeF cells, while virions produced by G355 cells contained the expected RD114 gp70. While the gp85 env-containing virions were infectious for permissive G355 cells, they were unable to infect FeF cells. The block to infection by the gp85-containing particles in FeF cells could be abrogated by treatment with the glycosylation inhibitor tunicamycin. Our results indicate that restriction of RD114 virus involves a novel mechanism dependent on two factors: altered glycosylation of the envelope to a gp85 form and an altered RD114 receptor in FeF cells.     

Evolution of the Retroviral Restriction Gene Fv1: Inhibition of Non-MLV Retroviruses

Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV). It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV). To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV) and feline foamy virus (FFV) respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.     

Homology-based identification of capsid determinants that protect HIV1 from human TRIM5α restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.     

Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

正Dear Editor,Previous studies had described the adaptation of enterovirus 71 (EV-A71) strains that enabled entry and viral replication in Chinese Hamster Ovary (CHO) cell line(Zaini and Mc Minn 2012; Zaini et al. 2012). These adapted     

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.     

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.     

A five-amino-acid deletion of the eastern equine encephalitis virus capsid protein attenuates replication in mammalian systems but not in mosquito cells

Eastern equine encephalitis virus (EEEV) is a human and veterinary pathogen that causes sporadic cases of fatal neurological disease. We previously demonstrated that the capsid protein of EEEV is a potent inhibitor of host cell gene expression and that this function maps to the amino terminus of the protein. We now identify amino acids 55 to 75, within the N terminus of the capsid, as critical for the inhibition of host cell gene expression. An analysis of stable EEEV replicons expressing mutant capsid proteins corroborated these mapping data. When deletions of 5 to 20 amino acids within this region of the capsid were introduced into infectious EEEV, the mutants exhibited delayed replication in Vero cells. However, the replication of the 5-amino-acid deletion mutant in C710 mosquito cells was not affected, suggesting that virus replication and assembly were affected in a cell-specific manner. Both 5- and 20-amino-acid deletion mutant viruses exhibited increased sensitivity to interferon (IFN) in cell culture and impaired replication and complete attenuation in mice. In summary, we have identified a region within the capsid protein of EEEV that contributes to the inhibition of host gene expression and to the protection of EEEV from the antiviral effects of IFNs. This region is also critical for EEEV pathogenesis.     

Retroviral capsid determinants of Fv1 NB and NR tropism

The specificity determinants for susceptibility to resistance by the Fv1 n and b alleles map to amino acid 110 of the murine leukemia virus CA protein. To study the interaction between Fv1 and CA, we examined changes in CA resulting in the loss of susceptibility to Fv1 resistance in naturally occurring NB- and NR-tropic viruses. A variety of amino acid changes affecting Fv1 tropism were identified, at CA positions 82, 92 to 95, 105, 114, and 117, and they all were mapped to the apparent exterior of virion-associated CA. These amino acids may form a binding surface for Fv1.     

The infectivity and lytic activity of minute virus of mice wild-type and derived vector particles are strikingly different

Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.