Klaus Wiegers

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.

Mouse-Human Heterokaryons Support Efficient Human Immunodeficiency Virus Type 1 Assembly

ABSTRACT 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.

N-AglB of poliovirus type 1: A discontinuous epitope formed by two loops of VP1 comprising residues 96–104 and 141–152

Analysis of resistant mutants to neutralizing monoclonal antibodies revealed a discontinuous neutralization epitope on VP1 of poliovirus type 1, Mahoney. The epitope has the unique property of being also part of a sequential epitope within neutralization antigenic site I (N-AgI). It is formed by residues in the loop 96-104 connecting the B and C strand and in the loop 141-152 connecting the D and E strand of VP1. Because of strong analogy to neutralization immunogen IB (NImIB) of human rhinovirus 14 (HRV-14) we have called this site N-AgIB of poliovirus type 1.

Activation of the glucocorticoid receptor releases unstimulated PBMCs from an early block in HIV-1 replication.

Infection of resting peripheral mononuclear blood cells (PBMCs) with HIV-1 is not productive due to a block prior to integration of the provirus into the host genome. Here we show that a unique restriction is determined by the status of the glucocorticoid receptor (GR). Proviral integration increases after addition of a GR ligand. The ligand dependent effect is confined to an early time period after infection and requires GR and the GR binding viral protein Vpr. Endogenous GR and transiently expressed Vpr are localized in the cytoplasm in unstimulated PMCs and comigrate into the nucleus upon ligand addition. Thus, the predominant cytoplasmic localization of GR seems to be a specific obstacle for HIV replication. Accordingly, efficient proviral integration in a cell line with a constitutive cytoplasmic GR requires addition of a GR ligand. The data suggest that steroids can overcome the restriction on HIV provirus formation and thereby increase the reservoir of virus producing cells.

A new antigenic site of poliovirus recognized by an intertypic cross-neutralizing monoclonal antibody

A monoclonal antibody (mAb 7J6) neutralizing poliovirus type 2 (PV2) and poliovirus type 1 (PV1) was obtained after immunization of BALB/c mice with infectious PV2, strain MEF-1. Preincubation of mAb 7J6 with PV1 inhibited its binding to PV2 and vice versa. Neutralization-resistant variants of PV2 and PV1 were selected. Nucleotide sequencing of the RNAs of some variants revealed mutations in the loop of amino acid residues 239 to 245 in VP2 and in the loop of amino acid residues 195 to 207 in VP3. This is the first evidence that these two loops contribute to a neutralization antigenic site (N-Ag) for poliovirus. Moreover, this new site on PV2 induced intertypic cross-neutralizing antibodies.