Biomedical subjects

H G Kräusslich

Publications and source records attributed to H G Kräusslich.

At least 19 recordsLinked to original sources

PubMed2001

The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated gene expression system, a novel monoclonal antibody, and in vitro transcription-translation were employed to investigate the subcellular localization and to characterize the membrane association of this viral protein. When expressed individually or in the context of the entire HCV polyprotein, NS4B was localized in the endoplasmic reticulum (ER), as shown by subcellular fractionation, immunofluorescence analyses, and double-label confocal laser scanning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred cotranslationally, presumably via engagement of the signal recognition particle by an internal signal sequence. In membrane extraction and proteinase protection assays NS4B displayed properties of a cytoplasmically oriented integral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. An efficient replication system will be essential to further define the role of NS4B in the viral life cycle.

Animals↗

PubMed2001

Nucleocytoplasmic RNA transport in retroviral replication.

Retroviral replication is highly dependent on post-transcriptional regulation because a single primary transcript directs synthesis of many viral proteins. The identification and characterization of two post-transcriptional regulatory systems (Rev/RRE and CTE) revealed the efficient use of cellular transport pathways by retroviruses to achieve production of infectious progeny virus. The Rev/RRE system of HIV-1 consists of the viral Rev protein which binds to its target sequence on incompletely spliced RNAs and channels these into the CRM1-dependent export pathway, which is normally used for export of cellular proteins and RNAs (U snRNAs and 5 S rRNA). The CTE, on the other hand, directly recruits the cellular mRNA export receptor TAP to the viral RNA. Both systems have in common that they recruit a key player of a specific cellular export pathway and this recruitment appears to out-compete the respective cellular target molecules. The fact that CTE can functionally substitute for Rev/RRE, yielding a replication-competent virus, indicates that very short sequence elements are sufficient for post-transcriptional control. The presence of short dominant export signals could relieve the selective pressure on the remainder of the genome to maintain a sequence that is easily exported. The resultant increase in permitted sequence space may increase the potential for immune escape, thereby providing a selective advantage for the virus. Replication of the CTE-dependent HIV-1 variant is significantly impaired compared with the wild-type virus. Considering that post-transcriptional control in the case of HIV is also used to provide a temporal switch from the early phase of regulatory protein expression to the late phase of virion production, one may suggest that the CRM1 export pathway is advantageous for the rapid delivery of large amounts of cargo (i.e. HIV RNA). This would be in accordance with its normal function because CRM1 has been shown to direct the nuclear export of cellular regulatory proteins which must be accomplished rapidly as well. In summary, retroviruses have evolved fascinating ways to deal with their cellular environment and to make use of cellular transport pathways, allowing nuclear export of intron-containing RNAs which are normally restricted to the nucleus. Specific signals on the viral RNAs recruit key factors of cellular export, thus bypassing these restrictions and ensuring efficient viral replication.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

A recombinant virus assay using full-length envelope sequences to detect changes in HIV-1 co-receptor usage.

The clinical management of HIV-1 infection has benefited enormously from molecular characterization of drug resistance as well as determination of the viral phenotype in vitro. HIV-1 infected individuals on HAART are currently monitored for the development of drug resistance variants allowing clinicians to redesign drug regimens. An understanding of the molecular basis of the evolution of drug resistance in vivo allows the improvement of the drugs as well as in vitro evaluation of new antiviral compounds alone or in combination with those currently approved. New findings suggest that viral envelopes could be a target to inhibit infection and replication. Therefore the generation of a recombinant virus assay (RVA) to allow the phenotypic determination of drug resistance against entry inhibitors (EI) is anticipated. We constructed an env-deleted clone of HIV-1 using the molecular clone NL-4.3. PCR amplified complete envelope genes (NL-4.3, BaL, primary envelope-genes) were ligated in vitro with a deletion clone (pNL-deltaK) and PM1-cells, supporting the replication of R5- and X4-tropic viruses, were transfected. Determination of co-receptor usage of the harvested recombinant virus-swarm revealed no difference compared to the molecular clones derived individually from three different patients. These results clearly show that an envelope-based RVA is practicable to monitor HIV-co-receptor usage at a given time point. Furthermore, this assay will allow to monitor resistance development against existing and future entry inhibitors and will aid to improve the management of HIV-therapy.

Antiretroviral Therapy, Highly Active↗

PubMed2001

Loss of N-linked glycans in the V3-loop region of gp120 is correlated to an enhanced infectivity of HIV-1.

We describe mutants of human immunodeficiency virus type-1 (HIV-1) strain NL4-3, which are lacking the thirteenth, fifteenth, or seventeenth sites for N-linked glycosylation (g13, g15, g17) of the envelope protein gp120. All three sites are located within the hypervariable V3 loop region of gp120. Those mutants lacking carbohydrates g15 or combinations of g15/g17 showed markedly higher infectivity for GHOST cells (human osteosarcoma cells) expressing CXCR4 (GHOST-X4), compared to the fully glycosylated NL4-3 wild type virus. In addition, these mutants could also infect cells which exhibits low background expression of CXCR4, corresponding to <10% of that observed for GHOST-X4 cells. In addition to the enhanced infectivity observed, mutants lacking g15 and g17 showed increased resistance to inhibition by SDF-1, the natural ligand of CXCR4. Thus, loss of the oligosaccharides g15 and g17 in the V3 region of gp120 markedly influences CXCR4-specific infection.

Amino Acid Sequence↗

PubMed2001

Organization of immature human immunodeficiency virus type 1.

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.

Capsid↗

PubMed2001

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression.

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.

Animals↗

PubMed2001

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

3T3 Cells↗

PubMed2000

Expression of human immunodeficiency virus type 1 Gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing HIV envelope.

Recombinant vesicular stomatitis viruses have been developed as high-level expression vectors which serve as effective vaccine vectors in animals (Roberts et al., 1998, J. Virol. 72, 4704-4711; Roberts et al., 1999, J. Virol. 73, 3723-3732). Here we show that two genes can be expressed simultaneously from a single, live-attenuated VSV recombinant. The genes used encode the Pr55(gag) protein precursor of HIV-1 (1.7-kb gene) and an HIV-1 envelope (Env) protein (2.4 kb gene). Our results show that VSV can accommodate up to a 40% increase in genome size with only a threefold reduction in virus titer. Recombinants expressing the Pr55(gag) protein precursor with or without Env protein produced abundant HIV virus-like particles (VLPs) in addition to bullet-shaped VSV particles. HIV Env protein expressed from a VSV recombinant also expressing Gag was specifically incorporated into the HIV VLPs but not into the VSV particles. In contrast, VSV G protein was found in both VSV particles and in HIV VLPs. Such VSV/HIV recombinants producing HIV VLPs with Env protein could be an effective source of HIV-like particles inducing both cellular and antibody-mediated immunity to HIV-1.

AIDS Vaccines↗

PubMed2000

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.

3' Untranslated Regions↗

PubMed2000

A conformational switch controlling HIV-1 morphogenesis.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV-1 Gag-derived protein can form spherical particles, virtually indistinguishable from immature HIV-1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV-1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C-terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

Animals↗

PubMed2000

Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors.

We present a systematic comparison of three modules that enhance expression from retroviral gene transfer vectors at a posttranscriptional level: (i) splice signals (SS) that create an intron in the 5' untranslated region; (ii) constitutive RNA transport elements (CTE), originally discovered in D-type retroviruses; and (iii) the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Here we show that enhancement of expression depends not only on the specific element, but also on the gene of interest, implying context-dependent activity of the RNA elements. Interestingly, different results were obtained for genes that normally require or do not require such control elements. Expression of the HIV-1 gag-protease gene, which normally depends on the viral export factor Rev, was strongly enhanced by an oligomeric CTE, while WPRE had only a marginal effect. On the other hand, both CTE and WPRE compensated for the lack of an intron in the expression of human beta-globin. In this case, the strongest stimulation of RNA production was observed when functional SS were combined with the WPRE. Both CTE and, in particular, WPRE also enhanced expression of cDNAs that do not normally require any such element (green fluorescent protein, human multidrug resistance-1). In this study, functional SS and WPRE acted in an additive manner, resulting in a 10-fold higher level of expression. Our results indicate that the described modules act on different levels of RNA processing, transport, and translation and that the correct choice of a posttranscriptional enhancer configuration depends on the type of cDNA to be expressed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Human immunodeficiency virus type 1 Vpr protein is incorporated into the virion in significantly smaller amounts than gag and is phosphorylated in infected cells.

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is a small accessory protein involved in the nuclear import of viral DNA and the growth arrest of host cells. Several studies have demonstrated that a significant amount of Vpr is incorporated into the virus particle via interaction with the p6 domain of Gag, and it is generally assumed that Vpr is packaged in equimolar ratio to Gag. We have quantitated the relative amount of Vpr in purified virions following [(35)S]cysteine labeling of infected MT-4 cells, as well as by quantitative immunoblotting and found that Vpr is present in a molar ratio of approximately 1:7 compared to capsid. Analysis of isolated core particles showed that Vpr is associated with the mature viral core, despite quantitative loss of p6 from core preparations. Metabolic labeling of infected cells with ortho[(32)P]phosphate revealed that a small fraction of Vpr is phosphorylated in virions and infected cells.

Capsid↗

PubMed2000

Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1.

Mature human immunodeficiency virus type 1 (HIV-1) particles contain a cone-shaped core structure consisting of the internal ribonucleoprotein complex encased in a proteinaceous shell derived from the viral capsid protein. Because of their very low stability after membrane removal, HIV-1 cores have not been purified in quantities sufficient for structural and biochemical analysis. Based on our in vitro assembly experiments, we have developed a novel method for isolation of intact mature HIV-1 cores. Concentrated virus suspensions were briefly treated with nonionic detergent and immediately centrifuged in a microcentrifuge for short periods of time. The resuspended pellet was subsequently analyzed by negative-stain and thin-section electron microscopy and by immunoelectron microscopy. Abundant cone-shaped cores as well as tubular and aberrant structures were observed. Stereo images showed that core structures preserved their three-dimensional architecture and exhibited a regular substructure. Detailed analysis of 155 cores revealed an average length of ca. 103 nm, an average diameter at the base of ca. 52 nm, and an average angle of 21.3 degrees. There was significant variability in all parameters, indicating that HIV cores are not homogeneous. Immunoblot analysis of core preparations allowed semiquantitative estimation of the relative amounts of viral and cellular proteins inside the HIV-1 core, yielding a model for the topology of various proteins inside the virion.

HIV-1↗

PubMed2000

Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines.

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.

Animals↗

PubMed2000

A block to human immunodeficiency virus type 1 assembly in murine cells.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.

3T3 Cells↗

PubMed1999

Toxins that are activated by HIV type-1 protease through removal of a signal for degradation by the N-end-rule pathway.

Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.

Amino Acid Sequence↗

PubMed1999

Competitive inhibition of human immunodeficiency virus type-1 protease by the Gag-Pol transframe protein.

The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.

Amino Acid Sequence↗