Retroviral pseudo-virus carrying the envelope proteins of neurotropic Friend murine leukemia virus effectively transferred retroviral vector into glial cells

We isolated the neurotropic Friend murine leukemia virus, FrC6 and its molecular clone A8, which proliferated in rat glial cell lines in vitro and in the rat brain in vivo. To investigate the contribution of viral envelope proteins to the neurotropism of A8 virus, the retroviral pseudo-virus carrying the envelope proteins of A8 virus and Moloney murine leukemia virus (MoMLV) was produced by transfecting the env gene of A8 virus (A8env) in the MoMLV based packaging cell, psi CRE. The phenotypically mixed pseudo-virus infected the rat glial cell lines as well as NIH 3T3 cells, whereas the psi CRE-produced pure pseudo-virus without A8env expression infected the glial cells at lower efficiency. Furthermore, the psi CRE cells with A8env expression produced pseudo-virus at a higher titer than normal psi CRE cells. The infectivity of the phenotypically mixed pseudo-virus to the glial cells was abolished by a neutralizing antibody against A8 virus, which did not reduce the ability of the psi CRE-produced pure pseudo-virus to infect NIH 3T3 cells. These results indicated that the envelope protein of A8 virus is assembled into the pseudo-viral particles and that it contributes to glial cell infection by the A8 virus.     

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.     

Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells

Human immunodeficiency virus type 1 (HIV-1) can readily accept envelope (Env) glycoproteins from distantly related retroviruses. However, we previously showed that the HIV-1 Env glycoprotein complex is excluded even from particles formed by the Gag proteins of another lentivirus, visna virus, unless the matrix domain of the visna virus Gag polyprotein is replaced by that of HIV-1. We also showed that the integrity of the HIV-1 matrix domain is critical for the incorporation of wild-type HIV-1 Env protein but not for the incorporation of a truncated form which lacks the 144 C-terminal amino acids of the cytoplasmic domain of the transmembrane glycoprotein. We report here that the C-terminal truncation of the transmembrane glycoprotein also allows the efficient incorporation of HIV-1 Env proteins into viral particles formed by the Gag proteins of the widely divergent Moloney murine leukemia virus (Mo-MLV). Additionally, pseudotyping of a Mo-MLV-based vector with the truncated rather than the full-length HIV-1 Env allowed efficient transduction of human CD4+ cells. These results establish that Mo-MLV-based vectors can be used to target cells susceptible to infection by HIV-1.     

Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins

Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.     

Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.     

Mechanisms for ganciclovir resistance in gastrointestinal tumor cells transduced with a retroviral vector containing the herpes simplex virus thymidine kinase gene

Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tumor cells confers sensitivity to the cells to the viral drug ganciclovir (GCV). Although the efficacy of the HSV-TK/GCV approach is well studied, the mechanisms for the resistance of HSV-TK-transduced tumor cells to GCV are poorly understood. Here, we examined the mechanisms for GCV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines. Our results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines. GCV-resistant colonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-resistant colonies demonstrated that the HSV-TK gene was either partially or completely deleted from the resistant HSV-TK-transduced cells. In the HT-29 RM and MIAPACA-2 RM cells, a 220-bp deletion of the gene was found, whereas in the HT-29 R1-R5-resistant cells, the whole TK gene was found to be absent. Immunocytochemical studies using a polyclonal antibody to the TK protein demonstrated that the HSV-TK protein was absent in the GCV-resistant, HSV-TK-transduced cells. Transfection of the resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV. The presence of GCV-resistant cells was only demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect. Our results suggest that GCV resistance found in tumor cells transduced with a retroviral HSV-TK gene is due to the lack of a functional TK protein in the tumor cells rather than any intrinsic resistance of the cells to GCV. In tumor cells with a good bystander effect, the small percentage of TK-transduced cells that do not express the TK protein are probably killed by the bystander effect because GCV-resistant tumor cells were not found in these cell lines. GCV-resistant tumor cells were found only in tumor cell lines with a poor bystander effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.     

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.     

Bacterial surface proteins recognized by CD4+ T cells during murine infection with Listeria monocytogenes

Optimal immunity to the Gram-positive pathogen Listeria monocytogenes (LM) requires both CD8+ and CD4+ antigen-specific T cell responses. Understanding how CD4+ T cells function in an immune response to LM and how bacterial proteins are processed to peptide/MHC class II complexes in infected cells requires identification of these proteins. Using LacZ-inducible, LM-specific CD4+ T cells as probes, we identified two immunogenic LM proteins by a novel expression cloning strategy. The antigenic peptides contained within these proteins were defined by deletion analysis of the genes, and their antigenicity was confirmed with synthetic peptides. The nucleotide sequences of the genes showed that they encode previously unknown LM proteins that are homologous to surface proteins in other bacterial species. Consistent with their surface topology, mild trypsin treatment of LM protoplasts ablated T cell recognition of these Ags. These findings establish a general strategy for identifying unknown CD4+ T cell Ags and demonstrate that LM surface proteins can provide the peptides for presentation by MHC class II molecules that are specific targets for CD4+ T cells during murine LM infection.     

Propentofylline inhibits production of TNFalpha and infection of LP-BM5 murine leukemia virus in glial cells

Much evidence points to the involvement of N-methyl-D-aspartate (NMDA) receptors in the development and maintainance of neuropathic pain. In neuropathic pain, there is generally involved a presumed opioid-insensitive component, which apparently can be blocked by NMDA receptor antagonists. However, in order to obtain complete analgesia, a combination of an NMDA receptor antagonist and an opioid receptor agonist is needed. Recent in vitro data have demonstrated that methadone, ketobemidone, and dextropropoxyphene, in addition to being opioid receptor agonists, also are weak noncompetitive NMDA receptor antagonists. Clinical anecdotes suggest that the NMDA receptor antagonism of these opioids may play a significant role in the pharmacological action of these compounds; however, no clinical studies have been conducted to support this issue. In the present commentary, we discuss evidence for the NMDA receptor antagonism of these compounds and its relevance for clinical pain treatment; an overview of structure-activity relationships for the relevant opioids as noncompetitive NMDA receptor antagonists also is given. It is concluded that although the finding that some opioids are weak noncompetitive NMDA receptor antagonists in vitro has created much attention among clinicians, no clinical studies have been conducted to evaluate the applicability of these compounds in the treatment of neuropathic pain conditions.     

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.     

Detection of a high frequency of virus-specific CD4+ T cells during acute infection with lymphocytic choriomeningitis virus

Lymphocytic choriomeningitis virus (LCMV), like many viruses, induces a profound activation and expansion of CD8+ T cells. In contrast, CD4+ T cells do not increase in total number during the acute infection. We show here that mice infected with LCMV have a low but detectable frequency (<1/300) of CD4+ T cells, as detected by IL-2 production in limiting dilution assays, to each of two class II peptides during the peak of the acute LCMV response and into long-term memory. However, during the peak of the acute CD4+ T cell response, >20% of the CD4+ T cells secreted IFN-gamma after stimulation with PMA and ionomycin, and >10% of the CD4+ T cells secreted IFN-gamma after stimulation with the LCMV peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound Ag-specific CD4+ T cell response during viral infections.     

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR-) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR- MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR- nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR- MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of approximately 45 A and a length of approximately 200 A. Because of the pleomorphic shape and paracrystalline packing of the Gag-RNA complexes, we raise the possibility that assembly of MuLV is driven by protein-RNA, as well as protein-protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.     

Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours

Replication-incompetent recombinant retroviruses are currently used for gene delivery. The limited efficiency of gene transfer using these vectors hampers implementation of gene therapy. Successful integration of Moloney murine leukemia virus (MMuLV)-derived retroviral vectors into the host cell DNA requires cell division. The time difference between virus entry and cell division is variable and prolonged in slowly dividing cells. Therefore, the rate of intracellular decay of internalized vectors between the time of entry into the target cell and cell division may limit the probability of successful integration following viral entry. We present two methods that measure the intracellular stability of MMuLV-derived retroviral vectors in NIH 3T3 cells. The first is based on a temporary interruption of cell cycle progression by using cell detachment. This method provides an estimate, but not a direct measurement, of the half-life. The results show that the MMuLV intracellular half-life is on the order of but shorter than the total cell cycle time. The second method allows the direct measurement of the intracellular half-life by using two cell cycle-specific labels: 5-bromodeoxyuridine, a thymidine analog that labels cells in S-phase; and the viral vector that labels cells in mitosis. By varying the time between the administration of the two labels, the intracellular half-life is measured to be in the range of 5.5 to 7.5 h. Such a short intracellular half-life may restrict the efficiency of gene transfer by retroviral vectors, particularly in slowly dividing target cells.     

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

Correlates of apoptosis of CD4+ and CD8+ T cells in tonsillar tissue in HIV type 1 infection

The immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased death by apoptosis of T cell subsets. In the present study, we have examined correlates of apoptosis of CD4+, CD8S+CD28+, and CD8+CD28- T cells in tonsillar lymphoid tissue in persons with HIV-1. Single-cell suspensions of tonsillar lymphocytes were analyzed by flow cytometry to determine the fraction of cells showing typical characteristics of apoptosis as well as the expression of activation markers within the live and the apoptotic cell populations. The proportion of cells carrying infectious provirus was quantified by limiting dilution analysis. Compared with uninfected controls, apoptosis of both CD4+ and CD8+ T cells was enhanced in HIV-1 infection and was higher among CD8+ than among CD4+ T cells. Apoptosis of CD28-cells was more prevalent than apoptosis of CD28+ cells for both CD4+ and CD8+ T cells. Occurrence of apoptosis of CD4+ T cells correlated with provirus levels and proportional expression of the activation marker HLA-DR. Apoptosis of CD8+CD28+ cells correlated with expression of the activation markers CD69 and HLA-DR while apoptosis within CD8+CD28- cells did not correlate with any of the studied parameters. Although apoptosis was much more prevalent among CD8+ than CD4+ T cells, CD8+ T cells still accumulated in tonsillar lymphoid tissue in persons with HIV-1. Our data may be interpreted to suggest that apoptosis of CD4+, CD8+CD28+, and CD8+CD28- cells in tonsillar tissue is regulated by different mechanisms and the results are of importance to our understanding of the immunopathogenesis of HIV-1 infection.     

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.