Receptor-Mediated Entry by Equine Infectious Anemia Virus Utilizes a pH-Dependent Endocytic Pathway

ABSTRACT Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.

Download Full-text

Equine Infectious Anemia Virus Entry Occurs through Clathrin-Mediated Endocytosis

Journal of Virology ◽

10.1128/jvi.01754-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1628-1637 ◽

Cited By ~ 20

Author(s):

Melinda A. Brindley ◽

Wendy Maury

Keyword(s):

Viral Entry ◽

Equine Infectious Anemia Virus ◽

Low Ph ◽

Endocytic Pathway ◽

Wild Type Virus ◽

Wild Type ◽

Coated Pits ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

In Cells

ABSTRACT Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAVvMA-1c, utilizes two mechanisms of entry. In cells such as ED cells that EIAVvMA-1c is able to superinfect, viral entry is pH independent and appears to be mediated by plasma membrane fusion, whereas in cells where no detectable superinfection occurs, EIAVvMA-1c entry that is low-pH dependent occurs through clathrin-coated pits in a manner similar to wild-type virus. Regardless of the mechanism of entry being utilized, the internalization kinetics of EIAV is rapid with 50% of cell-associated virions internalizing within 60 to 90 min. Cathepsin inhibitors did not prevent EIAV entry, suggesting that the low-pH step required by wild-type EIAV is not required to activate cellular cathepsins.

Download Full-text

Subpopulations of Equine Infectious Anemia Virus Rev Coexist In Vivo and Differ in Phenotype

Journal of Virology ◽

10.1128/jvi.77.22.12122-12131.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 12122-12131 ◽

Cited By ~ 21

Author(s):

Prasith Baccam ◽

Robert J. Thompson ◽

Yuxing Li ◽

Wendy O. Sparks ◽

Michael Belshan ◽

...

Keyword(s):

Complex Adaptive Systems ◽

Nuclear Export ◽

Adaptive Systems ◽

Equine Infectious Anemia Virus ◽

Environmental Changes ◽

Regulatory Protein ◽

Dependent Manner ◽

Equine Infectious Anemia ◽

Anemia Virus

ABSTRACT Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.

Download Full-text

The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Journal of Virology ◽

10.1128/jvi.72.10.8344-8348.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8344-8348 ◽

Cited By ~ 36

Author(s):

Feng Li ◽

Bridget A. Puffer ◽

Ronald C. Montelaro

Keyword(s):

Equine Infectious Anemia Virus ◽

Parental Virus ◽

Target Cells ◽

Monocyte Differentiation ◽

Cell Culture Systems ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

First Time

ABSTRACT Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and envgenes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAVUK. Various EIAVUK mutants lackingS2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAVUKmutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAVS2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.

Download Full-text

Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection

Journal of Virology ◽

10.1128/jvi.72.12.9612-9620.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 9612-9620 ◽

Cited By ~ 43

Author(s):

Wei Zhang ◽

Scott M. Lonning ◽

Travis C. McGuire

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Equine Infectious Anemia Virus ◽

Class I ◽

Target Cells ◽

Ctl Epitopes ◽

Peptide Epitopes ◽

Equine Infectious Anemia ◽

Anemia Virus

ABSTRACT Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.

Download Full-text

S2 from Equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3

10.1101/065078 ◽

2016 ◽

Author(s):

Ajit Chande ◽

Cristiana Cuccurullo ◽

Annachiara Rosa ◽

Serena Ziglio ◽

Susan Carpenter ◽

...

Keyword(s):

Equine Infectious Anemia Virus ◽

Critical Role ◽

Host Factor ◽

Target Cells ◽

Accessory Protein ◽

Post Translational Modification ◽

Virus Particles ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

Hiv 1

ABSTRACTThe lentivirus equine infectious anemia virus (EIAV) encodes S2, a pathogenic determinant important for virus replication and disease progression in horses. No molecular function has yet been linked to this accessory protein. We now report that S2 can replace the activity of Nef on HIV-1 infectivity, being required to antagonize the inhibitory activity of SERINC proteins on Nef-defective HIV-1. Similar to Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor AP2. Accordingly, a functional endocytic machinery is essential for S2-mediated infectivity enhancement, which is impaired by inhibitors of clathrin-mediated endocytosis. In addition to retargeting SERINC5 to a late endosomal compartment, S2 promotes the host factor degradation. Emphasizing the similarity with Nef, we show that S2 is myristoylated and, compatible with a crucial role of the post-translational modification, its N-terminal glycine is required for the anti-SERINC5 activity.EIAV-derived vectors devoid of S2 are less susceptible than HIV-1 to the inhibitory effect of both human and equine SERINC5. We then identified the envelope glycoprotein of EIAV as a determinant which also modulates retrovirus susceptibility to SERINC5, indicating a bi-modular ability of the equine lentivirus to counteract the host factor.S2 shares no sequence homology with other retroviral factors known to counteract SERINC5. Adding to primate lentivirus Nef and gammaretrovirus glycoGag, the accessory protein from EIAV makes another example of a retroviral virulence determinant which independently evolved SERINC5-antagonizing activity. SERINC5 therefore plays a critical role for the interaction of the host with diverse retrovirus pathogens.Significance StatementSERINC5 and SERINC3 are recently discovered cellular inhibitors of retroviruses, which are incorporated into virus particles and impair their ability to propagate the infection to target cells. Only two groups of viruses (represented by HIV-1 and MLV) have so far been identified to have evolved the ability of counteracting SERINC inhibition. We now discovered that Equine infectious anemia virus, which causes a debilitating disease in horses, also acquired the ability to protect the virus particle from inhibition by SERINC5 and SERINC3, using its small protein S2. The evidence that three different retroviruses have independently evolved the ability to elude inhibition bySERINC5 and SERINC3 indicates that these cellular factors play a fundamental role against various retrovirus pathogens.

Download Full-text

Cytotoxic T lymphocytes in protection against equine infectious anemia virus

Animal Health Research Reviews ◽

10.1079/ahr200482 ◽

2004 ◽

Vol 5 (2) ◽

pp. 271-276 ◽

Cited By ~ 14

Author(s):

Travis C. McGuire ◽

Darrilyn G. Fraser ◽

Robert H. Mealey

Keyword(s):

T Lymphocytes ◽

Mhc Class I ◽

Cytotoxic T Lymphocytes ◽

Equine Infectious Anemia Virus ◽

Neutralizing Antibody ◽

Target Cells ◽

T Helper ◽

Immunodeficiency Virus ◽

Equine Infectious Anemia ◽

Anemia Virus

AbstractCytotoxic T lymphocytes (CTL) are associated with virus control in horses infected with equine infectious anemia virus (EIAV). Early in infection, control of the initial viremia coincides with the appearance of CTL and occurs before the appearance of neutralizing antibody. In carrier horses, treatment with immunosuppressive drugs results in viremia before a change in serum neutralizing antibody occurs. Clearance of initial viremia caused by other lentiviruses, including human immunodeficiency virus-1 and simian immunodeficiency virus, is also associated with CTL and not neutralizing antibody. In addition, depletion of CD8+cells prior to infection of rhesus monkeys with simian immunodeficiency prevents clearance of virus and the same treatment of persistently infected monkeys results in viremia. Cats given adoptive transfers of lymphocytes from vaccinated cats were protected and the protection was MHC-restricted, occurred in the absence of antiviral humoral immunity, and correlated with the transfer of cells with feline immunodeficiency virus-specific CTL and T-helper lymphocyte activities. Therefore, a lentiviral vaccine, including one for EIAV, needs to induce CTL. Based on initial failures to induce CTL to EIAV proteins by any means other than infection, we attempted to define an experimental system for the evaluation of methods for CTL induction. CTL epitopes restricted by the ELA-A1 haplotype were identified and the MHC class I molecule presenting these peptides was identified. This was done by expressing individual MHC class I molecules from cDNA clones in target cells. The target cells were then pulsed with peptides and used with effector CTL stimulated with the same peptides. In a preliminary experiment, immunization of three ELA-A1 haplotype horses with an Env peptide restricted by this haplotype resulted in CTL in peripheral blood mononuclear cells (PBMC) which recognized the Env peptide and virus-infected cells, but the CTL response was transient. Nevertheless there was significant protection against clinical disease following EIAV challenge of these immunized horses when compared with three control horses given the same virus challenge. These data indicated that responses to peptides in immunized horses needed to be enhanced. Optimal CTL responses require help from CD4+T lymphocytes, and experiments were done to identify EIAV peptides which stimulated CD4+T lymphocytes in PBMC from infected horses with different MHC class II types. Two broadly cross-reactive Gag peptides were identified which stimulated only an interferon γ response by CD4+T lymphocytes, which indicated a T helper 1 response is needed for CTL stimulation. Such peptides should facilitate CTL responses; however, other problems in inducing protection against lentiviruses remain, the most significant of them being EIAV variants that can escape both CTL and neutralizing antibody. A possible solution to CTL escape variants is the induction of high-avidity CTL to multiple EIAV epitopes.

Download Full-text

Detection and Induction of Equine Infectious Anemia Virus-Specific Cytotoxic T-Lymphocyte Responses by Use of Recombinant Retroviral Vectors

Journal of Virology ◽

10.1128/jvi.73.4.2762-2769.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 2762-2769 ◽

Cited By ~ 15

Author(s):

S. M. Lonning ◽

W. Zhang ◽

S. R. Leib ◽

T. C. McGuire

Keyword(s):

Equine Infectious Anemia Virus ◽

Leukemia Virus ◽

Retroviral Vectors ◽

Retroviral Vector ◽

Simian Virus ◽

Target Cells ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

Ctl Responses

ABSTRACT Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.

Download Full-text

Endocytosis and a Low-pH Step Are Required for Productive Entry of Equine Infectious Anemia Virus

Journal of Virology ◽

10.1128/jvi.79.23.14482-14488.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14482-14488 ◽

Cited By ~ 24

Author(s):

Melinda A. Brindley ◽

Wendy Maury

Keyword(s):

Ammonium Chloride ◽

Equine Infectious Anemia Virus ◽

Cell Types ◽

Low Ph ◽

Host Cells ◽

Productive Infection ◽

Tissue Culture Cell ◽

Lysosomotropic Agents ◽

Equine Infectious Anemia ◽

Anemia Virus

ABSTRACT Recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-pH step. The entry mechanism of equine infectious anemia virus (EIAV) has yet to be examined. Here, we demonstrate that wild-type strains of EIAV require a low-pH step for productive entry. Lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of EIAV. The presence of ammonium chloride (30 mM), monensin (30 μM), or bafilomycin A (50 nM) in the medium dramatically decreased the number of EIAV antigen-positive cells. We found that a low-pH step was required for EIAV infection of tissue culture cell lines as well as primary cells, such as endothelial cells and monocyte-derived macrophages. The ammonium chloride treatment did not reduce virion stability, nor did the treatment prevent virion binding to cells. Consistent with a requirement for a low-pH step, virion infectivity was enhanced more than threefold by brief low-pH treatment following binding of viral particles to permissive cells. A superinfecting variant strain of EIAV, vMA-1c, did not require a low-pH step for productive infection of fibroblasts. However, lysosomotropic agents were inhibitory to vMA-1c infection in the other cell types that vMA-1c infected but did not superinfect, indicating that the entry pathway used by vMA-1c for superinfection abrogates the need for the low-pH step.

Download Full-text