Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

Structure and assembly of immature HIV.

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximately 17-A resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.

Chemogenetic ON and OFF switches for RNA virus replication.

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.

Genetic analysis and gene expression of human immunodeficiency virus.

Ten years after the initial description of acquired immune deficiency syndrome, its causative agent, the human immunodeficiency virus, remains the subject of intense scientific interest. Recent research has focused on the detailed analysis of the molecular principles governing gene expression and virion formation and on the cause of immune system dysfunction. Within the past year, considerable progress has been made regarding both the role of the regulatory proteins and the mechanism by which they function, and the determinants of cell tropism and of virion formation.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner.

Retroviral gene expression requires nuclear export and translation of incompletely spliced RNA. In the case of human immunodeficiency virus (HIV), this is facilitated by the viral Rev protein binding to its cognate RNA response element (RRE), while other retroviruses contain constitutive transport elements (CTE) binding to cellular factors. These CTE can substitute for the HIV-1 Rev/RRE system, albeit with reduced efficiency. Here, we show that multimeric copies of the CTE restore HIV-1 protein expression to levels comparable to or higher than Rev/RRE in various cell lines from different species. We suggest that multimerization of export factors is important for CTE function, as reported for Rev. CTE function was not affected when the element was displaced from its natural position close to the poly(A) signal, while insertion of an intron into the 3'-untranslated region (3'-UTR) severely reduced CTE activity. In this case, cytoplasmic RNA degradation was observed, which may be mediated by nonsense-mediated RNA decay. In contrast, Rev-dependent gene expression was insensitive to an intron in the 3'-UTR. Finally, we show that the putative CTE-binding protein RNA helicase A is not specifically translocated into the cytoplasm upon overexpression of CTE-containing RNA.

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

Polyprotein processing in picornavirus replication.

The primary translation product of the picornavirus genome is a single large protein which is processed to the mature viral polypeptides by progressive, co- and post-translational cleavages. Replication of the picornaviruses is thus entirely dependent upon the proteolysis of viral precursor proteins. In poliovirus, two virus-encoded proteinases have been identified that catalyze all but the final cleavage of the viral polyprotein. The final processing event, maturation of the virion polypeptide VPO, appears to occur by an unusual autocatalytic serine proteinase-like mechanism. Proteolytic processing of viral precursor proteins is basically similar in all picornaviruses, but recently it has become clear that there are also important differences between these viruses. Understanding of the processing events in picornavirus replication may ultimately lead to the discovery of specific inhibitors of the viral enzymes that could prove clinically useful as anti-viral agents.

Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24).

Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene. The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products. The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV. Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity. Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction. The precipitated CA was readily dissolved in low ionic strength aqueous buffer. Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form.

Coreceptor requirements of primary HIV type 1 group O isolates from Cameroon.

HIV-1 group O has its epicenter in Cameroon and neighboring countries and is responsible for 3 to 5% of all HIV infections in this region. It is believed that HIV-1 group O was introduced into the human population by a separate cross-species transmission, occurring independently of the HIV-1 (group M and group N) and HIV-2 transmissions. We have studied the coreceptor requirements of 12 primary HIV-1 O-type isolates from individuals with different clinical symptoms. Only 2 of these 12 viruses showed a syncytium-inducing phenotype after infection of primary peripheral blood mononuclear cells (PBMCs) and were infectious for the T cell line C8166. These isolates used CXCR4 as a coreceptor for entry, whereas the remaining isolates used only CCR5 efficiently. One isolate was able to use BOB and CCR8 as coreceptors in addition to CXCR4. All group O isolates tested were efficiently inhibited by SDF-1 or RANTES, the natural ligands of CXCR4 and CCR5, respectively. These results indicate that CXCR4 and CCR5 are the principal coreceptors for HIV-1 O-type viruses. Most of the HIV-1 group O isolates studied were derived from patients at later stages of the disease. Although HIV-1 group O and group M infections do not differ in their pathogenesis, the studied isolates did not evolve to use a broad range of coreceptors as described for HIV-1 group M and HIV-2.

Parameters influencing measurement of the Gag antigen-specific T-proliferative response to HIV type 1 infection.

We have analyzed factors that might influence the in vitro quantitation of the T-proliferative response to HIV-1 Gag antigens, a common and increasingly used clinical measurement of helper T cell function in the context of HIV-1 infection. We have compared the rate and extent of T cell proliferation in freshly prepared and previously frozen PBMC samples, and have concluded that frozen cells can be used successfully; we have assessed whether the suppression of any HIV-1 replication in the PBMC cultures affects the extent of T cell proliferation; we have studied which forms of the Gag antigens are the most efficient at inducing T cell proliferation. From the latter experiments, we conclude that Gag proteins that include p17, and perhaps also p7, sequences flanking the central p24 capsid protein, are better stimulants than proteins that comprise only p24 sequences.

Immunohematological reference values for healthy adults in Burkina Faso.

Reference ranges for peripheral blood lymphocyte subsets were generated for 186 healthy adults in Burkina Faso using single-platform flow cytometry. CD4(+) T-cell counts ranged from 631 to 1,696 cells microl(-1); they were lower in males (n = 97) than in females (n = 89), whereas natural killer cell counts were higher.

Evaluation of a simplified dual-platform flow cytometric method for measurement of lymphocyte subsets and T-cell maturation phenotypes in the population of Nouna, Burkina Faso.

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3(+) CD8(+) lymphocytes, and yields proportions of B cells and CD4(+) T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4(+) T cells (bias +/- precision, -1% +/- 6%) and CD8(+) T cells (-3% +/- 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean +/- standard deviation (SD) CD4(+)-to-CD8(+) T-cell ratio was 1.61 +/- 0.61, the mean percentage +/- SD of CD4(+) T cells was 42% +/- 7%, and that of CD8(+) T cells 29% +/- 7%. Among CD4(+) lymphocytes, 28% +/- 7% were classified as central memory (CD45RA(low) CCR7(+)), 22% +/- 10% as naïve (CD45RA(high) CCR7(+)), 45% +/- 12% as effector memory (CD45RA(low) CCR7(-)); and 5% +/- 3% as terminally differentiated effector memory expressing CD45RA (CD45RA(high) CCR7(-)). Among CD8(bright) lymphocytes, 3% +/- 2% had a central memory phenotype, 27% +/- 13% were naïve, 37% +/- 13% had an effector memory phenotype, and 34% +/- 12% were terminally differentiated effector memory cells expressing CD45RA.

Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity.

Enzymatically active retroviral proteinases are dimers of identical polypeptide chains with a fold similar to that of other aspartic proteinases. Each polypeptide chain, encoded on one of the viral polyproteins, is less than half the size of cellular aspartic proteinases and contains only one of the two active-site aspartate residues. A plasmid was constructed to generate a genetically linked dimer of the proteinase (PR) of human immunodeficiency virus (HIV) type 1, composed of two copies of the PR sequence linked by a structurally flexible hinge region. The expression product was stable and active against HIV polyprotein substrates. Mutational analysis revealed that the linked dimer, and not multimers thereof, contained the proteolytic activity. Expression of the linked dimer as a component of a HIV polyprotein by in vitro translation gave rapid autocatalytic processing, whereas the wild-type polyprotein was stable on prolonged incubation. Transfection of HIV subviral or proviral constructs, containing the linked dimer of HIV PR, gave premature processing of the viral polyproteins, thus preventing particle formation and infectivity. Premature processing also led to increased cell toxicity.

Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation.

The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Kräusslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959.

Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli.

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.

Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity.

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.