Packaging cell lines for simian foamy virus type 1 vectors

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.     

Ubiquitination of the human immunodeficiency virus type 1 env glycoprotein

Expression of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is stringently regulated in infected cells. The majority of the glycoprotein does not reach the cell surface but rather is retained in the endoplasmic reticulum or a cis-Golgi compartment and subsequently degraded. We here report that Env of various HIV-1 isolates is ubiquitinated at the extracellular domain of gp41 and that Env expression could be increased by lactacystin, a specific proteasome inhibitor, suggesting that the ubiquitin/proteasome system is involved in control of expression and degradation.     

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus env proteins possess a functionally conserved assembly domain.

The envelope (env) glycoproteins of human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) form dimers shortly after synthesis. Analysis of the simian immunodeficiency virus (SIV) env protein expressed by a recombinant vaccinia virus revealed that it, too, forms stable homodimers. When the HIV-1 and SIV env proteins or the HIV-1 and HIV-2 env proteins were coexpressed in the same cells, heterodimers were formed. Thus, the env proteins of HIV-1, HIV-2, and SIV possess a functionally conserved domain involved in subunit-subunit recognition and assembly that likely involves the ectodomain of gp41.     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.     

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.     

Characterization of a human lymphoblastoid cell line permanently modified by simian foamy virus type 10

Simian Spumavirinae serotype, SFV10, of a Papio cynocephalus baboon, was used to infect a human lymphoblastoid cell line, LV2. Permanent growth and morphological alterations of infected cells occurred, even though no viral particles were detected. Evidence for the presence of viral genomes in the modified cell line is provided indirectly from immunological studies and induction experiments followed by coculture procedures. The permanently modified cell line obtained (LV2-FB10) is an interesting model for the investigation of the possible integration of foamy viruses into the host genomes.     

Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus.

Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame.     

Semen-specific genetic characteristics of human immunodeficiency virus type 1 env

Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1.     

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.

The ability of poxviruses to undergo intramolecular recombination within tandemly arranged homologous sequences can be used to generate chimeric genes and proteins. Genes containing regions of nucleotide homology will recombine to yield a single sequence composed of portions of both original genes. A recombinant virus containing two genes with a number of conserved regions will yield a population of recombinant viruses containing a spectrum of hybrid sequences derived by recombination between the original genes. This scheme has been used to generate hybrid human immunodeficiency virus type 1 env genes. Recombinant vaccinia viruses that contain two divergent env genes in tandem array have been constructed. In the absence of selective pressure to maintain both genes, recombination between conserved homologous regions in these genes generated a wide range of progeny, each of which expressed a novel variant polypeptide encoded by the newly created hybrid env gene. Poxvirus-mediated recombination may be applied to map type-specific epitopes, to create novel pharmaceuticals such as hybrid interferons, to study receptor-binding or enzyme substrate specificities, or to mimic the antigenic diversity found in numerous pathogens.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.     

Identification of simian immunodeficiency virus SIVMAC env gene products.

A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.     

Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals.     

RNA of simian sarcoma-associated virus type 1 produced in human tumor cells.

Simian sarcoma-associated virus type 1 propagated in human rhabdomyosarcoma cells exhibited characteristics typical of oncornaviruses but seemed to have several aberrant properties. It had a buoyant density of 1.14 g/cm3, had RNA-dependent DNA polymerase activity, seemed to be labile to high salt concentrations, and contained little 50 to 60S RNA but relatively large amounts of human ribosomal RNA. In addition to 50 to 60S RNA, purified virions contained smaller RNA molecules with sedimentation coefficients of 28 to 30S, 18 TO 20S, and 4 to 10S. Unlike the 50 to 60S RNA species, the smaller virion-associated RNAs lacked polyadenylic acid, and the 28 to 30S RNA had an average base composition similar to that of human ribosomal RNA. Upon heat denaturation, the native 50 to 60S RNA genome yielded polyadenylic acid-containing 28 to 30S subunits that degraded in to 18 to 20S molecules upon further heat treatment. The 50 to 60S viral RNA had a guanine plus cytosine content of 56%.