High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.     

Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus.

Incorporation of human immunodeficiency virus type 1 (HIV-1) envelope proteins into vesicular stomatitis virus (VSV) particles was studied in a system that allows expressed envelope proteins to rescue phenotypically a temperature-sensitive mutant of VSV (tsO45). This mutant exhibits defective transport of its own envelope glycoprotein (G) and can be rescued by simultaneous expression of wild-type G protein from cDNA. We report here that a hybrid HIV-1-VSV protein containing the extracellular and transmembrane domains of the HIV-1 envelope protein fused to the cytoplasmic domain of VSV G protein was able to rescue the tsO45 mutant lacking the G protein, while the wild-type HIV-1 envelope protein was not. The VSV(HIV) pseudotypes obtained infected only CD4+ cells and were neutralized specifically by anti-HIV-1 sera. Our results indicate that the cytoplasmic tail of the VSV glycoprotein contains an independent signal capable of directing a foreign protein into VSV particles. The VSV(HIV) pseudotypes generated here were prepared in the absence of HIV-1 and should be useful for identifying molecules that block HIV-1 entry.     

Incorporation of high levels of chimeric human immunodeficiency virus envelope glycoproteins into virus-like particles

The human immunodeficiency virus (HIV) envelope (Env) protein is incorporated into HIV virions or virus-like particles (VLPs) at very low levels compared to the glycoproteins of most other enveloped viruses. To test factors that influence HIV Env particle incorporation, we generated a series of chimeric gene constructs in which the coding sequences for the signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) domains of HIV-1 Env were replaced with those of other viral or cellular proteins individually or in combination. All constructs tested were derived from HIV type 1 (HIV-1) Con-S DeltaCFI gp145, which itself was found to be incorporated into VLPs much more efficiently than full-length Con-S Env. Substitution of the SP from the honeybee protein mellitin resulted in threefold-higher chimeric HIV-1 Env expression levels on insect cell surfaces and an increase of Env incorporation into VLPs. Substitution of the HIV TM-CT with sequences derived from the mouse mammary tumor virus (MMTV) envelope glycoprotein, influenza virus hemagglutinin, or baculovirus (BV) gp64, but not from Lassa fever virus glycoprotein, was found to enhance Env incorporation into VLPs. The highest level of Env incorporation into VLPs was observed in chimeric constructs containing the MMTV and BV gp64 TM-CT domains in which the Gag/Env molar ratios were estimated to be 4:1 and 5:1, respectively, compared to a 56:1 ratio for full-length Con-S gp160. Electron microscopy revealed that VLPs with chimeric HIV Env were similar to HIV-1 virions in morphology and size and contained a prominent layer of Env spikes on their surfaces. HIV Env specific monoclonal antibody binding results showed that chimeric Env-containing VLPs retained conserved epitopes and underwent conformational changes upon CD4 binding.     

Production of High-Titer Human Immunodeficiency Virus Type 1 Pseudotyped with Vesicular Stomatitis Virus Glycoprotein

We describe a method for the production of high-titer stocks of human immunodeficiency virus type 1 (HIV-1) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV G). VSV G pseudotypes provide several advantages over other retroviral envelope proteins. The VSV G envelope is mechanically stable, enabling ultracentrifugal concentration of virions to high titers, and VSV G has a broad host range, enabling infection of many mammalian and nonmammalian cell types. VSV G pseudotypes of HIV-1 are useful for the study of HIV infection and replication kinetics and for the study of the function of specific viral proteins. We describe applications for the study of HIV-1 using VSV G pseudotypes. Additionally, we describe a method for pseudotyping retroviral vectors with VSV G. The same advantages of VSV G pseudotypes of HIV-1 apply to retroviral vectors; VSV G pseudotyped retroviral vectors may be used to introduce genes of interest into a wide variety of cell lines.     

Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.     

Induction of anti-HIV-1 immune responses by retroviral vectors.

Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.     

Separable mechanisms of attachment and cell uptake during retrovirus infection

In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1) gag and pol, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.     

The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor?

The Vpu protein is a human immunodeficiency virus type 1 (HIV-1)-specific accessory protein that is required for the efficient release of viral particles from infected cells. Even though HIV-2 does not encode Vpu, we found that this virus is nevertheless capable of efficiently releasing virus particles. In fact, the rate of virus release from HeLa cells transfected with a full-length molecular clone of HIV-2, ROD10, was comparable to that observed for the vpu+ HIV-1 NL4-3 isolate and was not further enhanced by expression of Vpu in trans. However, consistent with previous observations showing that HIV-2 particle release is Vpu responsive in the context of HIV-1/HIV-2 chimeric constructs; exchanging the gag-pol region of NL4-3 with the corresponding region from pROD10 rendered the resulting chimeric virus Vpu responsive. Our finding that the responsiveness of HIV-2 particle release to Vpu is context dependent suggested the presence of a Vpu-like factor(s) encoded by HIV-2. Using chimeric proviruses encoding HIV-2 gag and pol in the context of the HIV-1 provirus that were coexpressed with subgenomic HIV-2 constructs, we found that the HIV-2 envelope glycoprotein had the ability to enhance HIV-2 particle release with an efficiency comparable to that of the HIV-1 Vpu protein. Conversely, inactivation of the HIV-2 env gene in the original ROD10 clone resulted in a decrease in the rate of viral particle release to a level that was comparable to that of Vpu-deficient HIV-1 isolates. Providing the wild-type envelope in trans rescued the particle release defect of the ROD10 envelope mutant. Thus, unlike HIV-1, which encodes two separate proteins to regulate virus release or to mediate viral entry, the HIV-2 Env protein has evolved to perform both functions.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.     

Development of targeted gene transfer into human primary T lymphocytes and macrophages using high-titer recombinant HIV vectors

Primary human lymphocytes and macrophages are an important target cells for human immunodeficiency virus (HIV). For targeted gene transfer into CD4(+) lymphocytes and macrophages, we constructed HIV vectors with envelope glycoprotein (gp120) from the T-cell tropic BH10 strain and the macrophage tropic SF162, and developed an improved strategy for preparation of high-titer HIV vectors. Among several possible procedures, we found that ultrafiltration using CENTRIPREP columns was highly effective to concentrate HIV particles. The titer could be increased four orders of magnitudes. The total recovery was more than 80%. No replication-competent cytopathic HIV was detected in concentrated vector preparation. Using the high-titer HIV vector carrying the enhanced green fluorescent protein (EGFP) gene, we transduced human primary lymphocytes and macrophages. FACS analysis showed that the T-cell tropic vector could transduce 40-80% of CD4(+) T-cells stimulated with IL2 plus PHA and 20-50% of unstimulated cells. The macrophage tropic vector was shown to transduce approximately 20% of terminally differentiated macrophages. These results represent the initial report of targeted gene transfer into terminally differentiated macrophages. These results also indicate that these HIV vectors are useful for the manipulation of gene expression in HIV infectable cells and the development of gene therapy targeting lymphocytes and macrophages.     

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.     

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.     

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.     

Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity.

The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry.     

Redirecting retroviral tropism by insertion of short, nondisruptive peptide ligands into envelope

A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.     

HIV—1核蛋白p24在昆虫细胞中的表达

将完整的ＨＩＶ－１ ｐ２４基因克隆到杆状病毒转移质粒中,使用重组转移质粒与野生型杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经筛选获得带有编码ｐ２４基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中表达了ＨＩＶ核蛋白ｐ２４。其重组蛋白的分子量为２４ｋＤ。此重组糖蛋白在免疫荧光,免疫印染和酶联免疫实验中都能被人ＨＩＶ－１阳性血清和单克隆抗体所识别。     

Virion-Targeted Viral Inactivation of Human Immunodeficiency Virus Type 1 by Using Vpr Fusion Proteins

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4⁺ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4⁺ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6^gag domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.