Generation of a Recombinant Gag Virus-Like-Particle Panel for the Evaluation of p24 Antigen Detection by Diagnostic HIV Tests

Background

Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.

Methods

We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4^th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.

Results

Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4^th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.

Conclusions

The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.     

Direct phenotypical and functional dysregulation of primary human B cells by human immunodeficiency virus (HIV) type 1 in vitro

Background

Human immunodeficiency virus type 1 (HIV-1) induces a general dysregulation of immune system. Dysregulation of B cell compartment is generally thought to be induced by HIV-related immune activation and lymphopenia. However, a direct influence of HIV-1 particles on B cells was recently proposed as the third pathway of B cells dysregulation.

Methods/Principal Findings

We evaluated the direct and specific consequences of HIV-1 contact on activation, survival, proliferation and phenotype of primary B cells in vitro. Moreover, we examined expression of activation-induced cytidine deaminase (AID) mRNA that is responsible for class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that changes observed in cellular proliferation, phenotypes and activation of B cells could be caused by direct contact between HIV-1 particles and primary B cells in vitro. Finally, direct HIV-1-derived B cells activation led to the increase of AID mRNA expression and its subsequent CSR function was detected in vitro.

Conclusion/Significance

We showed that HIV-1 could directly induce primary B cells dysregulation triggering phenotypical and functional abilities of B cells in vitro that could explain in some extent early B-cell abnormalities in HIV disease.     

Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1-derived lentiviral vectors

Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.     

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background

Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.

Methodology/Principal Findings

Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.

Conclusions/Significance

These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.     

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2

Background

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.

Methodology/Principal Findings

L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.

Conclusion/Significance

VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.     

Influenza virus-like particles containing M2 induce broadly cross protective immunity

Background

Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health.

Methodology/Principal Findings

To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms.

Conclusions/Significance

These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.     

Human Immunodeficiency Virus Type 2 Vpx-Gag Interaction

Incorporation of Vpx into human immunodeficiency virus type 2 (HIV-2) virus-like particles is mediated by the Gag polyprotein. We have identified residues 15 to 40 of Gag p6 and residues 73 to 89 of Vpx as being necessary for virion incorporation. In addition, we show enhanced in vitro binding of Vpx to a chimeric HIV-1/HIV-2 Gag construct containing residues 2 to 49 of HIV-2 p6 and demonstrate that the presence of residues 73 to 89 of Vpx allows for in vitro binding to HIV-2 Gag.     

Lentivirus vectors using human and simian immunodeficiency virus elements

Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1-derived vectors is their ability to infect nondividing cells. Although high-titer HIV-1-derived vectors have been produced, concerns regarding safety still exist. Safety concerns arise mainly from the possibility of recombination between transfer and packaging vectors, which may give rise to replication-competent viruses with pathogenic potential. We describe a novel lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SIVpack/G. In this system, an HIV-1-derived genome is encapsidated by SIVmac core particles. These core particles are pseudotyped with VSV glycoprotein G. Because the nucleotide homology between HIV-1 and SIVmac is low, the likelihood of recombination between vector elements should be reduced. In addition, the packaging construct (SIVpack) for this lentivirus system was derived from SIVmac1A11, a nonvirulent SIV strain. Thus, the potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. To our knowledge, these experiments constitute the first demonstration that the HIV-1-derived genome can be packaged by an SIVmac capsid. We demonstrate that the lentivirus vector described here recapitulates the biological properties of HIV-1-derived vectors, although with increased potential for safety in humans.     

Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles

Background

Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles.

Methodology/Principal Findings

Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10⁶-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity.

Conclusions/Significance

This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.     

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.     

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.     

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity

Background

HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14⁺ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.

Methodology/Principal Findings

We analyzed sialoadhesin expression on CD14⁺ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14⁺ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-α and interferon-γ but not tumor necrosis factor-α. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.

Conclusions/Significance

Increased sialoadhesin expression on CD14⁺ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14⁺ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.     

Human immunodeficiency virus type 1 modified to package Simian immunodeficiency virus Vpx efficiently infects macrophages and dendritic cells

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.     

The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells

Background

Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before.

Methodology/Principal Findings

Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA.

Conclusion

Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.