Substitution of Yor1p NBD1 residues improves the thermal stability of Human Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508. To investigate substitutions that can stabilize this domain, we constructed chimeras between human CFTR and its closest yeast homolog Yor1p. The α-subdomain of Yor1p was replaced with that of CFTR in Saccharomyces cerevisiae. Cellular localization of green fluorescence protein-tagged Yor1p-CFTR chimeras was analyzed by fluorescence microscopy and quantitative multispectral imaging flow cytometry, steady-state protein levels were compared by SDS-PAGE and protein function probed by a phenotypic oligomycin resistance assay. The chimeras exhibited ER retention in yeast characteristic of defective protein folding/processing. Substitution of seven CFTR α-subdomain residues that are highly conserved in Yor1p and other transporters but differ in CFTR (S495P/R516K/F533L/A534P/K536G/I539T/R553K) improved Yor1p-CFTR chimera localization to the yeast plasma membrane. When introduced into human CFTR expressed in mammalian cells, the same substitutions improve the purified protein thermal stability. This stabilized human CFTR protein will be directly useful for structural and biophysical studies that have been limited by the thermal sensitivity of wild type CFTR. The insights into critical structural residues within CFTR could facilitate development of effective therapeutics for CF-causing mutations.

Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV.

Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c+ DC subsets were identified: CD11b+ and CD11blow DCs. CD11b+CD14+ DCs were the most abundant throughout the tract. A majority of CD11c+CD14+ cells corresponded to CD1c+ myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103+ DCs, located exclusively in the endometrium, whereas DC-SIGN+ DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14+ DC-SIGN+ as well as CD14+ DC-SIGN- cells captured virus, with ∼30% of these cells representing CD1c+ myeloid DCs. CD103+ DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.

Moloney murine leukemia virus integrase protein augments viral DNA synthesis in infected cells.

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Safety considerations in vector development.

The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5' sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the psi packaging signal joined with an open gag coding region. Analysis of the 3' end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector design prevents the generation of env-minus recombinant lentivirus containing a functional gag-pol structure (LTR-gag-pol-LTR), which is absolutely required for retroviral DNA mobilization and the emergence of RCR. Quality assurance based on monitoring for RCR may have limitations as a predictor of safety in vivo, especially in the long term. The demonstration of lentivirus infection via alternative entry mechanisms supports this notion. Therefore, the approach of monitoring trans-vector stocks for env-minus recombinant virus in vitro as a surrogate marker for the possible emergence of RCR in vivo should represent a significant advancement in vector safety quality assurance.

Analysis of lenti- and trans-lentiviral vector genetic recombination.

Lentiviral vectors hold great promise for gene therapy, and clinical trials to examine their safety and efficacy for treating human disease are being planned. The principle concern for safety is that genetic recombination among components of the vector could lead to the emergence of replication competent retrovirus (RCR). Using a sensitive method for detecting genetic recombination, we found that the current design of lentiviral vectors permits the generation of envelope-deficient recombinant lentivirus, stable integration of the recombinant into chromosomes of transduced cells, and mobilization of the recombinant genomes to other cells when pseudotyped with an exogenous envelope. We split the lentiviral packaging construct (Gag/Gag-Pol) into two separate parts: one that expresses Gag and Gag-Pro, and another that expresses Pol (reverse transcriptase [RT] and integrase [IN]) as a fusion partner of Vpr (Vpr-RT-IN). This "trans-lentiviral" vector efficiently transduces non-dividing cells and achieves titres greater than 10(6) U/ml or 10(8) IU/ml after concentration by ultracentrifugation. The trans-lentiviral vector disarms the Gag-Pol structure and prevents the generation of recombinants containing functional RT and IN. Since RT and IN are absolutely required for any type of RCR and DNA mobilization, this new class of lentiviral vector, in combination with our sensitive in vitro assay for monitoring regeneration of the gag-pol structure, offers a unique advantage for predicting vector safety for clinical applications.

Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiated mice.

Lentiviral vectors efficiently transduce human CD34(+) cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1(+)c-Kit(+)Lin(-) bone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120.

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC(50)) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log(10) higher than the mean IC(50) for CXCR4 (X4) isolates (P = 0. 0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC(50) for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log(10) higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC(50) for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log(10) lower than the IC(50) for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

Development of a novel trans-lentiviral vector that affords predictable safety.

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) hold great promise for gene therapy. However, the possibility of generating replication-competent retrovirus (RCR) through genetic recombination raises concerns for safety. Here we describe a novel HIV-based packaging system (trans-lentiviral) that splits gag/gag-pol into two parts: One that expresses gag/gag-pro and another that expresses reverse transcriptase and integrase as fusion partners of viral protein R (Vpr). Using a sensitive assay developed to specifically detect recombinant lentiviral DNA mobilization, we demonstrated that the trans-lentiviral vector prevents the generation of recombinants that contain a functional gag-pol structure, while the lentiviral vector generates env-minus recombinant lentivirus that mobilizes recombinant genomes to other cells when pseudotyped with an exogenous envelope. Since an intact gag-pol structure is absolutely required for retroviral DNA mobilization and RCR, the trans-lentiviral vector design significantly reduces this risk. Moreover, it makes it possible to assess the risk of RCR and DNA mobilization using an in vitro assay that monitors trans-lentiviral vector stocks for the regeneration of the gag-pol structure. Therefore, the trans-lentiviral vector design will ensure the greatest predictable level of safety for the clinical application of retroviral vectors, including HIV-based vectors.

Analysis of human immunodeficiency virus type 1 containing HERV-K protease.

The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.

Targeting human immunodeficiency virus (HIV) type 2 integrase protein into HIV type 1.

Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell's chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3' processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN(2)) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN(2) into IN mutant HIV-1 virions by expression in trans as a Vpr-IN fusion protein. Trans-complementation with IN(2) markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126-2135, 1999), cells infected with IN(2)-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, the trans-IN(2) protein was shown to support integration up to a level of 55% compared with that of the homologous trans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex.

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

The cysteine-cysteine family of chemokines RANTES, MIP-1alpha, and MIP-1beta induce trypanocidal activity in human macrophages via nitric oxide.

This paper describes a new role for the cysteine-cysteine (CC) chemokines RANTES, MIP-1alpha, and MIP-1beta on human macrophage function, which is the induction of nitric oxide (NO)-mediated trypanocidal activity. In a previous report, we showed that RANTES, MIP-1alpha and MIP-1beta enhance Trypanosoma cruzi uptake and promote parasite killing by human macrophages (M. F. Lima, Y. Zhang, and F. Villalta, Cell. Mol. Biol. 43:1067-1076, 1997). Here we study the mechanism by which RANTES, MIP-1alpha, and MIP-1beta activate human macrophages obtained from healthy individuals to kill T. cruzi. Treatment of human macrophages with different concentrations of RANTES, MIP-1alpha, and MIP-1beta enhances T. cruzi trypomastigote phagocytosis in a dose peak response. The optimal response induced by the three CC chemokines is attained at 500 ng/ml. The macrophage trypanocidal activity induced by CC chemokines can be completely inhibited by L-N-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine:NO pathway, but not by its D-enantiomer. Culture supernatants of chemokine-treated human macrophages contain increased NO2- levels, and NO2- production is also specifically inhibited by L-NMMA. The amount of NO2- induced by these chemokines in human macrophages is comparable to the amount of NO2- induced by gamma interferon. The killing of trypomastigotes by NO in cell-free medium is blocked by an NO antagonist or a NO scavenger. This data supports the hypothesis that the CC chemokines RANTES, MIP-1alpha, and MIP-1beta activate human macrophages to kill T. cruzi via NO, which is an effective trypanocidal mechanism.

Incorporation of functional human immunodeficiency virus type 1 integrase into virions independent of the Gag-Pol precursor protein.

Retroviral integrase (IN) is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. IN catalyzes integration of the proviral DNA into host cell chromosomes during the early stages of the virus life cycle, and as a component of Gag-Pol, it is involved in virion morphogenesis during late stages. It is unknown whether the scheme, conserved among retroviruses, for expressing and incorporating IN as a component of the Gag-Pol precursor protein is necessary for its function in the infected cell after viral entry. We have developed human immunodeficiency virus (HIV) virion-associated accessory proteins (Vpr and Vpx) as vehicles to deliver both foreign and viral proteins into the virus particle by their expression in trans as heterologous fusion proteins (X. Wu, et al., J. Virol. 69:3389-3398, 1995; X. Wu, et al., J. Virol. 70:3378-3384, 1996; X. Wu, et al., EMBO J. 16:5113-5122, 1977). To analyze IN function independent of its expression as a part of Gag-Pol, we expressed and incorporated IN into HIV type 1 (HIV-1) virions in trans as a fusion partner of Vpr (Vpr-IN). Our results demonstrate that the Vpr-IN fusion protein is efficiently incorporated into virions and then processed by the viral protease to liberate the IN protein. Virus derived from IN-minus provirus is noninfectious. However, this defect is overcome by trans complementation with the Vpr-IN fusion protein. Moreover, complemented virions are able to replicate through a complete cycle of infection, including formation of the provirus (integration). These results show, for the first time, that full IN function can be provided in trans, independent of its expression and incorporation into virions as a component of Gag-Pol. This finding also indicates that the IN domain of Gag-Pol is not required for the formation of infectious virions when IN is provided in trans. The ability to incorporate functional IN into retroviral particles in trans will provide unique opportunities to explore the function of this critical enzyme in a biologically relevant context, i.e., in infected cells as part of the nucleoprotein/preintegration complex.

Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein.

The expression and incorporation of retroviral enzymes into virions in the form of Gag/Pol precursor polyproteins is believed to be important for the assembly of infectious viral particles. HIV-1 encodes a 160 kDa Gag/Pol precursor that includes Gag, protease (PR), reverse transcriptase (RT) and integrase (IN). We have developed the use of HIV accessory proteins (Vpr and Vpx) as vehicles to incorporate protein of both viral and non-viral origin into virions by expression in trans as heterologous fusion proteins (Wu et al., 1995, 1996a). To analyze the role of Gag/Pol in the formation of infectious virions, we incorporated RT and IN into HIV-1 particles in trans, as fusion partners of viral protein R (Vpr). Virions derived from an RT and IN minus proviral clone were infectious and replicated through a complete cycle of infection when RT and IN proteins were provided in trans. These results demonstrate that functional RT and IN proteins can be provided in trans, and that their expression and incorporation into virions as components of Gag/Pol are not required for the formation of infectious virions. Thus, for the first time, we have demonstrated for a human pathogenic retrovirus that processes of assembly and the function of critical viral enzymes can be unlinked. This finding will provide unique opportunities to explore retroviral RT/IN function and the role of Gag/Pol in the formation of infectious virions in the context of a replicating virus (in vivo).

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.

Proteolytic activity of human immunodeficiency virus Vpr- and Vpx-protease fusion proteins.

In addition to Gag, Pol, and Env, primate lentiviruses encode other virion-associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressed in trans with HIV proviruses (Wu et al., J. Virol. 69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIV PR coding region was fused in-frame with the HIV-2 vpx and HIV-1 vpr genes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gag demonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles. Trans-expression of a PR-Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gag precursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr arguments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function.

The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.

Assessment of antiretroviral therapy by plasma viral load testing: standard and ICD HIV-1 p24 antigen and viral RNA (QC-PCR) assays compared.

To assess the utility of quantitative competitive-polymerase chain reaction (QC-PCR) measurements of plasma human immunodeficiency virus type 1 (HIV-1) RNA and other viral load markers for assessment of antiretroviral therapy, we used archived cryopreserved specimens from a randomized controlled clinical trial of 135 patients (CD4+ T cell count < or = 500/mm3), comparing zidovudine (500 mg/day) versus the nonnucleoside reverse transcriptase inhibitor L-697, 661 (50, 300, or 1,000 mg daily). We evaluated treatment-associated changes in plasma viral load by standard and immune complex-dissociated (ICD) HIV-1 p24 antigen assays, and, in a representative subset of patients (n = 46), by QC-PCR determination of virion-associated HIV-1 RNA. At baseline, HIV-1 RNA was quantifiable by QC-PCR in all patients tested (100%), whereas standard and ICD HIV-1 p24 antigen tests were positive (> or = 30 pg/ml) in 42% and 56%, respectively. All viral load parameters showed significant decreases from baseline within 1 week of initiation of zidovudine, as measured by standard p24 antigen assay, ICD p24 assay, and QC-PCR. At 1 week, patients treated with either 300 or 1,000 mg/day of L-697,661 showed significant decreases from baseline in plasma standard and ICD p24 antigen and QC-PCR-determined HIV-1 RNA levels. Whereas viral load decreases seen with zidovudine were sustained for the duration of treatment, plasma viral markers often returned to pretreatment levels despite ongoing L-697,661 treatment, with evidence of the emergence of drug-resistant virus. Whereas standard p24, ICD p24, and viral RNA levels changed similarly in response to treatment, the superior sensitivity and available dynamic range of plasma viral RNA assays like QC-PCR analysis provide an advantage for clinical monitoring of plasma viral load, allowing tracking of treatment-related changes even in patients with earlier stage disease and lower levels of viral load.

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.

Polyclonal rabbit antisera that detect the Vpr protein of SIVSM and SIVMAC on immunoblots of purified virions.

Antisera suitable for detection of SIVSM or SIVMAC Vpr proteins on Western blots of purified virions are currently not available. We have expressed the Vpr protein of SIVSMPBj1.9 in a gst-based prokaryotic expression system and used it to raise polyclonal antisera in rabbits. Two immune sera were obtained that specifically recognized both cell- and virion-associated Vpr protein on immunoblots of three different SIV isolates (SIVSMPBj1.9, SIVMACBK28, and SIVMAC239). Because Vpr is believed to play an important role in HIV/SIV replication and pathogenesis, these reagents will allow the extension of functional analyses of this protein to a broader spectrum of viruses. Both antisera and the gst-Vpr expression plasmid have been submitted to the NIAID AIDS Research and Reagent Program and are available to interested investigators.