Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function.

Repression of the endogenous human papillomavirus (HPV) type 18 E7 gene in HeLa cervical carcinoma cells by the bovine papillomavirus E2 transcription factor activates the retinoblastoma (Rb) pathway and induces cells to undergo senescence. To determine whether activation of the Rb pathway is responsible for senescence in response to HPV18 E7 repression, we tested the ability of wild-type and mutant E7 proteins to affect the activity of the Rb pathway and to modulate senescence in these cells. Enforced expression of the wild-type HPV16 E7 protein prevented Rb activation in response to E2 expression and impaired senescence. Importantly, there was an absolute correlation between the ability of mutant E7 proteins to inactivate the Rb pathway and to inhibit senescence in HeLa cells. Similar results were obtained in HT-3 cervical carcinoma cells. These results provide strong genetic evidence that activation of the Rb pathway is required for senescence in response to E7 repression. Hence, continuous neutralization of the Rb pathway by the E7 protein is required to maintain the proliferation of cervical carcinoma cells. Similarly, our results indicate that activation of the Rb pathway can prevent apoptosis induced by repression of the HPV18 E6 gene in HeLa cells.

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context.T he chemical diversity of the 20 standard amino acid sidechains found in proteins supports myriad biochemical activities essential for life, and additional chemical diversity is generated by posttranslational amino acid modifications. The number of potential protein sequences is enormous: for proteins only 300 amino acids long, ∼10 400 possible sequences exist, a number larger by many orders-of-magnitude than the number of atoms in the known universe. This immense number of sequences and the chemical and conformational complexity of naturally occurring proteins hinder our ability to understand protein structure, folding, and function, and complicate protein engineering efforts. In addition, many different related amino acid sequences can fold into nearly identical structures, and even proteins with quite divergent sequences or protein folds can use similar chemistry to carry out related functions or display the same catalytic activity (e.g., refs. 1-3). These considerations further complicate attempts to clearly identify and understand the key structural features of proteins. Thus, the isolation of biologically active proteins with drastically reduced chemical complexity would represent a significant advance in protein science by facilitating the correlation of specific structural features with function.Up to 30% of all proteins contain ∼20-25-amino acid, primarily hydrophobic segments that span cell membranes (4). These transmembrane domains often play critical roles in cellular processes by engaging in highly specific protein-protein and protein-lipid interactions required for the proper folding, oligomerization, and function of transmembrane proteins (5-8). For example, receptor tyrosine kinases (RTKs) such as the PDGF-β receptor (PDGFβR) usually consist of an extracellular ligand binding do...

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.transmembrane protein complex | oncogene | traptamer | BPV | blue native gel electrophoresis B ecause viruses modulate signaling nodes that control cell behavior and virus replication, the study of viral proteins has provided great insight into many aspects of cellular biochemistry and the cellular processes that regulate biological function. Thus, viral proteins have long been recognized as valuable tools to probe central problems in biology. A particularly interesting viral protein is the 44-residue E5 oncoprotein encoded by bovine papillomavirus type 1 (BPV). The BPV E5 protein and closely related E5 proteins of other fibropapillomaviruses are the shortest known, naturally occurring proteins with tumorigenic potential (1). The E5 protein is an extremely hydrophobic integral membrane protein located primarily in the membranes of the Golgi apparatus of transformed cells (2, 3). Biophysical studies in model membranes indicate that the E5 protein adopts a transmembrane orientation roughly perpendicular to the membrane surface (4-6). In essence, the E5 protein is a freestanding transmembrane domain (TMD), with a type II transmembrane orientation in which a short C-terminal segment protrudes into the lumen of the Golgi (2). In cells, detergent micelles, and model lipid bilayers, the E5 protein exists as a homodimer stabilized by disulfide bonds involving C-terminal cysteine residues (3-5, 7-10). Genetic studies showed that E5 dimerization is required for transforming activity, and a preferred orientation of the E5 dimer with a symmetric homodimer interface has been identified (7,9,11,12).The E5 protein transforms cells by activating the cellular platelet-derived growth factor (PDGF) β receptor (PDGFβR), although there may be additional minor, alternative transforming pathways as well (13). The PDGFβR is a receptor tyrosine kinase (RTK) with an extracellular domain that binds PDGF, a hydrophobic membrane-spanning segment, and a cytoplasmic domain with tyrosine kinase activity. The inactive PDGFβR is primarily monomeric in unstimulated cells, and PDGF binding indu...

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