Construction and characterization of recombinant VLPs and Semliki-Forest virus live vectors for comparative evaluation in the SHIV monkey model

For testing of recombinant virus-like particles (VLPs) in the SHIV monkey model, SIVmac239 Pr56gag precursor-based pseudovirions were modified by HIV-1 gp160 derived peptides. First, well-characterized epitopes from the HIV-1 envelope glycoprotein were inserted into the Pr56gag precursor by replacing defined regions that were shown to be dispensable for virus particle formation. Expression of these chimeric proteins in a baculovirus expression system resulted in efficient assembly and release of non-infectious, hybrid VLPs. In a second approach the HIV-1IIIB external glycoprotein gp120 was covalently linked to an Epstein-Barr virus derived transmembrane domain. Coexpression of the hybrid envelope derivative with the Pr56gag precursor yielded recombinant SIV derived Pr56gag particles with the HIV-1 gp120 firmly anchored on the VLP surface. Immunization of rhesus monkeys with either naked VLPs or VLPs adsorbed to alum induced substantial serum antibody titers and promoted both T helper cell and cytotoxic T lymphocyte responses. Furthermore, priming macaques with the corresponding set of recombinant Semliki-Forest viruses tended to enhance the immunological outcome. Challenge of the immunized monkeys with chimeric SHIV resulted in a clearly accelerated reduction of the plasma viremia as compared to control animals.     

Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain

It is now well accepted that the structural protein Pr55(Gag) is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55(Gag) is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55(Gag) sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55(Gag)-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development.     

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles

The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.     

Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus

We have used a recombinant vaccinia virus (VV) which expresses high levels of human immunodeficiency virus-1 (HIV-1) gag proteins to analyze the processing pathway of the gag p55 precursor. HIV-1 gag proteins were isolated from [3H]leucine-labeled VV:gag-infected H9 T lymphocytes by immunoprecipitation with either anti-p24, anti-p17, or anti-p6 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that processing of the p55 precursor involves three major intermediates (p41a, p41b, and p39). The p41a and p39 proteins contain the p17 and p24 protein segments, and the p41b is comprised of p24 and p15 segments. On two-dimensional gels, each intermediate as well as the mature p24 and p17 proteins migrated as distinct species. [3H]Myristic acid labeling of the HIV-1 gag proteins revealed that in addition to p55 and p17, the p41a and p39 intermediates, but not p41b, are myristylated, confirming that myristylation occurs at the NH2 terminus before cleavage of the p55 precursor protein. We conclude that the myristylated HIV-1 gag p55 precursor is initially cleaved at random either at the p17/p24 junction or at two sites between p24 and p15 proteins, resulting in three intermediates (p41a, p41b, and p39) which are subsequently cleaved to yield mature gag proteins.     

Formation of virus-like particles by HIV-1 Gag proteins, expressed by a recombinant vaccinia virus

Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.     

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.     

The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a virion-associated regulatory protein. Mutagenesis has shown that the virion association of Vpr requires sequences near the C terminus of the HIV-1 Gag polyprotein Pr55gag. To investigate whether Vpr incorporation is mediated by a specific domain of Pr55gag, we examined the ability of chimeric HIV-1/Moloney murine leukemia virus (MLV) Gag polyproteins to direct the incorporation of Vpr. Vpr expressed in trans did not associate with particles formed by the authentic MLV Gag polyprotein or with particles formed by chimeric Gag polyproteins that had the matrix (MA) or capsid (CA) domain of MLV precisely replaced by the corresponding domain of HIV-1HXB2. By contrast, Vpr was efficiently incorporated upon replacement of the C-terminal nucleocapsid (NC) domain of the MLV Gag polyprotein with HIV-1 p15 sequences. Vpr was also efficiently incorporated into particles formed by a MLV Gag polyprotein that had the HIV-1 p6 domain fused to its C terminus. Furthermore, a deletion analysis revealed that a conserved region near the C terminus of the p6 domain is essential for Vpr incorporation, whereas sequences downstream of the conserved region are dispensable. These results show that a virion association motif for Vpr is located within residues 1 to 46 of p6.     

Contribution of virus-like particles to the immunogenicity of human immunodeficiency virus type 1 Gag-derived vaccines in mice

The human immunodeficiency virus type 1 (HIV-1) Gag protein is a major target antigen for cytotoxic-T-lymphocyte-based vaccine strategies because of its high level of conservation. The murine model has been used extensively to evaluate potential HIV-1 vaccines. However, the biology of HIV-1 Gag is somewhat different in human and murine tissues. The ability of HIV-1 Gag to form virus-like particles (VLPs) in human cells is severely curtailed in murine cells. Hence, it is not known whether immunizing mice with expression vectors encoding HIV-1 Gag provides an accurate assessment of the immunogenicity of these candidate vaccines in primates. In this report, we made use of a chimeric Moloney murine leukemia virus (MMLV)-HIV-1 Gag in which the p17 matrix domain of HIV-1 was replaced with the p15 matrix and p12 domains from MMLV. Murine cells expressing this construct released significant amounts of VLPs. The construct preserved H-2d-restricted antigenic determinants in the remaining portion of HIV-1 Gag, allowing immunogenicity studies to be performed with mice. We demonstrated that immunizing mice with plasmid DNA or adenoviral vectors encoding this chimeric Gag did not significantly increase the HIV-1 Gag-specific cellular or humoral immune response when compared to immunization with a myristoylation-incompetent version of the construct. Thus, the release of VLPs formed in vivo may not play a major role in the immunogenicity of vectors expressing HIV-1 Gag constructs.     

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition.     

Spatial configuration of hepatitis E virus antigenic domain

Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T=1 virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T=1 HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope.     

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.     

Blocking of human immunodeficiency virus type-1 virion autolysis by autologous p2(gag) peptide

Our previous study suggested that the p2(gag) peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2(gag) peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2(gag) peptide (100 and 250 micro M) resulted in a decrease in the amount of p24(gag )in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2(gag) fusion peptide was synthesized to effectively deliver the p2(gag) peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV-1 cells, as detected on indirect immunofluorescence analysis using anti-p2(gag) peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2(gag) peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24(gag )in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2(gag) peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.