Heather L. Greenstone

Chimeric Papillomavirus-like Particles

We have constructed chimeric papillomavirus-like particles (CVLPs) by replacing the 34-carboxy-terminal amino acids of the HPV 16 L1 protein with various parts of the HPV 16 E7 protein. Chimeric proteins were expressed by recombinant baculoviruses and analyzed by electron microscopy for their ability to assemble into virus capsids. We were able to produce CVLPs in high efficiencies with inserts of up to 60 amino acids. CVLPs are able to induce a neutralizing antibody response, assayed by inhibition of hemagglutination of mouse erythrocytes. CVLPs are interacting with the putative receptor for papillomaviruses as they were shown to hemagglutinate mouse red blood cells and bind to and penetrate cells in vitro. As CVLPs follow a similar intracellular pathway as observed earlier for BPV VLPs, we speculate that CVLPs can be used to deliver peptides into mammalian cells in vitro and in vivo, possibly reaching the pathway for MHC class I presentation.

Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution

The three-dimensional structure of bovine papillomavirus has been determined to 9 Å resolution by reconstruction of high resolution, low dose cryo-electron micrographs of quench-f rozen virions. Although hexavalent and pentavalent capsomeres form star-shaped pentamers of the major capsid protein L1, they have distinct high-resolution structures. Most prominently, a 25 Å hole in the centre of hexavalent capsomeres is occluded in the pentavalent capsomeres. This raises the possibility that the L2 minor capsid protein is located in the centre of the pentavalent capsomeres. Inter-capsomere connections ∼10 Å in diameter were clearly resolved. These link adjacent capsomeres and are reminiscent of the helical connections that stabilize polyomavirus.

Human Herpesvirus 6 and Measles Virus Employ Distinct CD46 Domains for Receptor Function

We employed a quantitative cell fusion assay to identify structural domains of CD46 required for its function as a receptor for human herpesvirus 6 (HHV-6). We examined the activities of recombinant variants of CD46, including different isoforms as well as engineered truncations and molecular chimeras with decay-accelerating factor, a related protein in the family of regulators of complement activation (RCA). We observed strong receptor activity for all four CD46 isoforms, which differ in the membrane-proximal extracellular and cytoplasmic domains, indicating that the critical determinants for HHV-6 receptor activity reside outside the C-terminal portion of CD46. Analysis of the short consensus repeat (SCR) regions that comprise most of the extracellular portion of CD46 indicated a strong dependence on SCRs 2 and 3 and no requirement for SCRs 1 or 4. Fusion-inhibition studies with SCR-specific monoclonal antibodies supported the essential role of SCRs 2 and 3 in HHV-6 receptor activity. These findings contrast markedly with fusion mediated by measles virus glycoproteins for which we observed a strict dependence on SCRs 1 and 2, consistent with previous reports. These results expand the emerging notion that CD46 and other members of the RCA family are co-opted in distinct manners by different infectious pathogens.

L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

ABSTRACT BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

CD46 on glial cells can function as a receptor for viral glycoprotein‐mediated cell–cell fusion

Membrane cofactor protein (CD46) is a regulator of complement activation that also serves as the entry receptor for human herpes virus 6 (HHV‐6) and measles virus (MV) into human cells. While it is clear that oligodendrocytes and astrocytes are cell types commonly infected by these viruses, it is unclear whether oligodendrocytes express CD46, or which are the cellular mechanisms underlying the infection. We show that adult oligodendrocytes, as well as astrocytes and microglial cells, express CD46 on the cellular surface. Moreover, we employed a quantitative fusion assay to demonstrate that HHV‐6A infection of T lymphocytes enables cell–cell fusion of these cells to astrocytes or to oligodendroglial cells. This fusion is mediated by the interaction between viral glycoproteins expressed on the membrane of the infected cells and CD46 on the glial targets, and is also observed using cells expressing recombinant MV glycoproteins. These data suggest a mechanism that involves cell–cell fusion by which certain viruses could spread the infection from the periphery to the cells in the nervous system. Published 2005 Wiley‐Liss, Inc.

NIAID resources for developing new therapies for severe viral infections

Abstract Severe viral infections, including hemorrhagic fever and encephalitis, occur throughout the world, but are most prevalent in developing areas that are most vulnerable to infectious diseases. Some of these can also infect related species as illustrated by the threatened extinction of gorillas by Ebola infection in west and central Africa. There are no safe and effective treatments available for these serious infections. In addition to the logistical difficulties inherent in developing a drug for infections that are sporadic and occur mainly in the third world, there is the overwhelming barrier of no hope for return on investment to encourage the pharmaceutical industry to address these unmet medical needs. Therefore, the National Institute of Allergy and infectious Disease (NIAID) has developed and supported a variety of programs and resources to provide assistance and lower the barrier for those who undertake these difficult challenges. The primary programs relevant to the development of therapies for severe viral infections are described and three case studies illustrate how they have been used. In addition, contact information for accessing these resources is supplied.