Expression Of Env Protein Research Articles

Use of retroviral vectors for the analysis of SIV/HIV-specific CD8 T cell responses

CD8 + T cell responses and particularly cytotoxic T lymphocyte (CTL) activity are critical factors in controlling SHIV, SIV or HIV replication during natural infection and represent key parameters which need to be monitored during vaccine development. In order to improve the methodology for measuring CD8 + T cell responses, retroviral vectors expressing the full-length SIV-Gag or HIV-Env proteins were constructed and used to transduce B lymphoblastoid cell lines (BLCL) from cynomolgus monkeys infected with SHIV89.6P. Continuous expression of Gag and Env proteins was detected in stably transduced BLCL, which induced Gag- or Env-specific T cell responses, as measured by both IFNγ−ELISPOT and chromium release assays, upon in vitro stimulation of PBMC from the SHIV89.6P-infected monkeys. Moreover, induction of Gag-specific CTL using BLCL transduced with retroviral vector expressing the SIV-Gag protein was more efficient and specific compared to that obtained using BLCL infected with a recombinant vaccinia virus (rVV) encoding for the same antigen. Assays on purified CD4 + and CD8 + T cells indicated that both populations specifically produced IFNγ, but only the CD8 + T cells mediated Gag- and Env-specific cytotoxicity, indicating preferential expansion of these effector cells. Thus, this method represents an alternative tool for the analysis of CTL responses during vaccination protocols in those animal models where little information is available on MHC class I alleles or CTL epitopes.

N-Linked glycosylation is required for XC cell-specific syncytium formation by the R peptide-containing envelope protein of ecotropic murine leukemia viruses.

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R(+) Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R(+) Env protein but not that induced by the R(-) Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R(+) Env protein. The R(+) Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R(+) Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R(+) Env protein.

Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation.

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.

Contribution of the rev gene to the immunogenicity of DNA vaccines targeting the envelope glycoprotein of HIV.

The Rev protein of HIV plays a critical role in the export of viral mRNA from the nucleus to the cytoplasm of infected cells. This work examines the effect of introducing rev into a DNA vaccine encoding the Env protein of HIV, and compares the activity of env genes regulated by CMV versus CAG promoters. The HIV Env gp160 encoding gene with or without the rev gene was subcloned into a CMV promoter or a CAG promoter-driven expression plasmid. The Env protein expression of the plasmids was examined in vitro and the HIV-specific immunity was explored in BALB/c mice by an intramuscular route. The immune mice were intraperitoneally challenged with an HIV Env-expression vaccinia virus. Results indicate that the CAG promoter induces significantly higher levels of Env expression, and better immune responses, than the CMV promoter. Incorporating the rev gene into these plasmids further boosts antigen expression and immunogenicity. Indeed, vaccination with the pCAGrev/env or pCMVrev/env plasmid resulted in 1000-fold lower viral load than that with pCMVenv when the mice were challenged with an Env-expressing vaccinia virus. Incorporating rev into a DNA vaccine significantly increases the level of expression and immunogenicity of a co-expressed env gene, and that protective efficacy is further improved by utilizing a pCAG promoter.

A Glycosylation-Defective Variant of the Ecotropic Murine Retrovirus Receptor Is Expressed in Rat XC Cells

The XC rat sarcoma cell line undergoes extensive cell-to-cell fusion (syncytium formation) after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope (Env) protein in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R+ Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To assess whether this property of the XC cell line arises from differences in its ecotropic MLV receptor, CAT1, we isolated CAT1 cDNA clones from XC cells. A variant CAT1 (xcCAT1) was found together with the wild-type rat CAT1 (rCAT1). xcCAT1 cDNA encodes a protein with a single amino acid change that destroys a conserved N-linked glycosylation site proximal to the Env-binding motif. We found that xcCAT1 expressed in Chinese hamster ovary (CHO) cells undergoes less glycosylation than rCAT1 and that the expression of xcCAT1 rendered the CHO cells more susceptible to infection with Moloney MLV. Thus, N-glycosylation negatively regulates the receptor activity of rCAT1. This is supported by the observation that treatment of rat F10 cells with the N-glycosylation inhibitor tunicamycin enhanced their susceptibility to Mo-MLV. However, xcCAT1-expressing CHO cells did not fuse with 293T cells expressing R+ Env, indicating that xcCAT1 expression is not sufficient to induce the XC cell-specific characteristic of forming syncytia in response to R+ Env.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.

An evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity.

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K(959) in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.

Assembly and Release of SIV Env Proteins with Full-Length or Truncated Cytoplasmic Domains

We have used recombinant vaccinia viruses expressing full-length or truncatedgagorenvgenes of SIVmac239 to investigate the requirements for assembly of SIV proteins. We observed that assembly of virus-like particles (VLPs) was found to be 3- to 5-fold higher with full-length Env than with the truncated forms, or than VLPs containing only Gag proteins, in primary monkey cells or various human cell lines. When cells expressing Env proteins in the absence of Gag were examined by immunoelectron microscopy, clusters of Env protein and membrane vesicles containing Env proteins were observed at cell surfaces. A low level of vesicles was released from cells expressing full-length Env, but about a 10-fold higher level was released in cells expressing a truncated form of Env [Env733(t)] in which the cytoplasmic domain is only 17 amino acids in length. Another truncated protein, Env718(t), with a short cytoplasmic tail of 3 aa, was also incorporated into VLPs at a 10-fold higher level than the full-length Env protein and was more efficiently released in vesicles. The mature SU and TM proteins were predominantly incorporated into VLPs with full-length Env, but both cleaved and uncleaved precursor proteins were present in VLPs with truncated Env as well as in Env and Env(t) vesicles. A more prominent layer of spikes was seen by electron microscopy in VLPs with truncated Env than in VLPs containing full-length Env. These results indicate that truncated Env proteins have the ability to self-associate on the cell surface and are assembled into a more closely packed array than full-length Env, which could explain the preferential incorporation of Env proteins with short cytoplasmic tails into virions.

Structure and Expression of Endogenous Retroviral Sequences in the Permanent LMH Chicken Cell Line

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev 1 is present in duplicate whereas the locus corresponding to ev 3 is present in one copy. The other loci are probably ev 6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev 3 and ev 6 transcripts but no ev 1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

Expression of HERV-K ENV proteins

Expression of HERV-K ENV proteins

Normal T cell receptor-mediated signaling in T cell lines stably expressing HIV-1 envelope glycoproteins.

A series of T cell lines transfected to stably express HIV-1 envelope (env) glycoproteins were analyzed for viability and for T cell signaling. One transfectant was distinguished by its stable expression of gp120 and gp41, whereas the remainder of the T cell lines were similar to previously reported env-expressing T cells in synthesizing predominantly unprocessed env glycoprotein gp160. All of the transfectants were additionally constructed to express tat and rev proteins. None of these cell lines displayed growth abnormalities or spontaneous cell fusion, although the cell line synthesizing env gp120/gp41 could be induced to fuse and die when cocultured with a second cell expressing surface CD4. A cell line expressing only gp160 and the transfectant expressing gp160, gp120, and gp41 could be triggered normally via CD3-cross-linking as measured by protein tyrosine phosphorylation and by the induction of the CD69 activation marker. At levels of env protein expression sufficient to mediate syncytium formation and to kill cells, these HIV-1 env transfectants displayed no intrinsic T cell signaling abnormalities, suggesting that mechanisms other than a direct intracellular action of the tat or env proteins may be contributing to the deficit in Ag-specific T cell activation described subsequent to HIV infection in vivo and in vitro.

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.

Mechanism of Translation of Monocistronic and Multicistronic Human Immunodeficiency Virus Type 1 mRNAs

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.

Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs.

The effect of Rev on cytoplasmic accumulation of the singly spliced human immunodeficiency virus type 1 (HIV-1) vif, vpr, and env/vpu RNAs was examined by using a quantitative RNA polymerase chain reaction (PCR) analysis following transfection of complete proviral molecular clones into lymphoid cells. Previously published studies using subgenomic env constructs in nonlymphoid cell types concluded that Rev was necessary for cytoplasmic accumulation of high levels of unspliced env RNA and that, by analogy, Rev must be necessary for the cytoplasmic accumulation of all HIV-1 RNAs that contain the Rev-responsive element (RRE). We confirm those results in COS cells. Unexpectedly, in lymphoid cells, we find that although Rev acts somewhat to increase the cytoplasmic level of full-length HIV-1 RNA, Rev has little or no effect on cytoplasmic accumulation of singly spliced HIV-1 RNAs. However, Env protein expression was greatly reduced in the absence of Rev. Analysis of the cytoplasmic RNA revealed that in the absence of Rev or the RRE, the cytoplasmic vif, vpr, and env/vpu 2 RNAs were not associated with polysomes but with a complex of 40S-80S in size. Consequently, efficient expression of the Vif, Vpr, Vpu, and Env proteins from these RNAs is dependent on Rev. These results exclude a mechanism whereby the sole function of Rev is simply to export RNAs from nucleus to cytoplasm. We discuss other models to take into account the dependence on Rev for efficient translation of cytoplasmic HIV-1 RNAs.

Generation and characterization of the human immunodeficiency virus type 1 mutants

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.

A retroviral gp70-related protein is expressed at specific stages during mouse oocyte maturation and in preimplantation embryos

Mouse monoclonal antibodies directed against different epitopes of the murine leukemia virus envelope protein gp70 were used to study the expression of retroviral related env proteins during mouse oocyte maturation, fertilization and blastocyst formation. A gp70-related antigen was detected with immunohistochemistry in growing oocytes but not in primordial and primary non-growing oocytes. Atretic oocytes were also negative. Both parthenogenetically activated and fertilized oocytes were positive. Two-cell stages showed a patchy distribution of the antigen. Later preimplantation stages were negative except for a markedly positive period during compaction of the morula and adhesion of the blastocyst.

Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins.

Gene expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by two trans-acting factors encoded by the pX region, p40tax and p27tax.p40tax is a transcriptional activator and p27rex is a post-transcriptional regulator. Using full-length viral DNA, we studied the regulatory effects of rex on HTLV-1 gene expression. p27rex is required for expression of both gag and env proteins, increasing the level of their mRNAs. The effect was dependent on the dose of p27rex expression plasmid. In parallel, increased doses of p27rex suppressed the expression of fully spliced pX mRNA, which encodes the regulatory proteins. These two effects of p27rex operated at the post-transcriptional level and were independent of transcriptional regulation. Lowering the level of pX mRNA down-regulates transcription of the proviral genome. These observations demonstrate that rex is a positive post-transcriptional regulator for gag, pol and env protein expression, and acts at the same time as an indirect negative regulator of viral transcription.