Reinhold Welker

Organization of Immature Human Immunodeficiency Virus Type 1

ABSTRACT Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particles 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.

Biochemical and Structural Analysis of Isolated Mature Cores of Human Immunodeficiency Virus Type 1

ABSTRACT 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°. 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.

Membrane-Anchored Peptide Inhibits Human Immunodeficiency Virus Entry

ABSTRACT Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

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

ABSTRACT 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 novelcis-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 polopen 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.

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.