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Biomedical subjects

Edward M Campbell

Publications and source records attributed to Edward M Campbell.

7 recordsLinked to original sources
PubMed2006

Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell.

GFP-Vpr labeled HIV-1 virions have provided a method to visually examine the interactions between the virus and target cell during infection. However, existing methods to discriminate between virions that have been non-specifically endocytosed from those that have productively entered the host cell cytoplasm have remained problematic. Therefore, we examined the ability of a series of membrane-targeted fluorescent fusion protein constructs to be incorporated into virions. We find that a fluorescent protein fusion targeted to the plasma membrane by the addition of the N-terminal 15 amino acid sequence of c-Src (S15) is efficiently packaged into HIV virions. Using fluorescent proteins fused to this sequence, we have generated virions dually labeled with S15-mCherry and GFP-Vpr. Importantly, we can detect the loss of this S15-mCherry membrane signal following fusion. After infection with VSV-g pseudotyped HIV virions, we find a measurable, specific loss of membrane label during infection. This loss of fluorescence is not observed when fusion is prevented using bafilomycin A. This increased ability to discriminate between non-productively endocytosed virions and those actively undergoing steps in the infectious process will facilitate efforts to examine early steps in infection microscopically.

Cell Line↗
PubMed2006

Proteasome inhibitors uncouple rhesus TRIM5alpha restriction of HIV-1 reverse transcription and infection.

The primate TRIM5alpha proteins have recently been defined as cellular restriction factors, preventing primate infection by retroviruses from different species. For instance, rhesus TRIM5alpha (rhTRIM5alpha) restricts infection by HIV-1. Virtually all TRIM5alpha proteins block the early replication of retroviruses by preventing the accumulation of reverse transcription products, but the underlying mechanism remains unclear. In this article, we find that disrupting proteasome function alters rhTRIM5alpha localization and allows the normal generation of HIV-1 late reverse transcription products, even though HIV-1 infection and the generation of nuclear 1-LTR and 2-LTR viral cDNA forms remain impaired. This finding suggests rhTRIM5alpha restricts HIV infection in two distinct phases: (i) altering the normal passage of the reverse-transcribing viral genome to the nucleus and (ii) targeting the reverse transcription complex to be disrupted by the proteasome. Because proteasome inhibitor blocks the second phase, accumulation of a nonfunctional viral DNA genome can be readily observed. Defining each phase may reveal HIV-1 targets for future antiviral therapy in which dual blockade may be equally as effective as naturally occurring rhTRIM5alpha protein in preventing HIV-1 infection in vivo.

Animals↗
PubMed2006

Proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse TRIM5 proteins.

The primate TRIM5 proteins constitute a class of restriction factors that prevent host cell infection by retroviruses from different species. The TRIM5 proteins act early after virion entry and prevent viral reverse transcription products from accumulating. We recently found that proteasome inhibitors altered the rhesus monkey TRIM5alpha restriction of human immunodeficiency virus type 1 (HIV-1), allowing reverse transcription products to accumulate even though viral infection remained blocked. To assess whether sensitivity to proteasome inhibitors was a common feature of primate TRIM5 proteins, we conducted a similar analysis of restriction mediated by owl monkey TRIM-cyclophilin A (CypA) or human TRIM5alpha. Similar to rhesus monkey TRIM5alpha restriction, proteasome inhibition prevented owl monkey TRIM-CypA restriction of HIV-1 reverse transcription, even though HIV-1 infection and the output of 2-LTR circles remained impaired. Likewise, proteasome inhibition alleviated human TRIM5alpha restriction of N-tropic murine leukemia virus reverse transcription. Finally, HIV-1 reverse transcription products escaping rhesus TRIM5alpha restriction by proteasome inhibition were fully competent for integration in vitro, demonstrating that TRIM5alpha likely prevents the viral cDNA from accessing chromosomal target DNA. Collectively, these data indicate that the diverse TRIM5 proteins inhibit retroviral infection in multiple ways and that inhibition of reverse transcription products is not necessary for TRIM5-mediated restriction of retroviral infection.

Animals↗
PubMed2004

Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity.

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.

Actins↗
PubMed2003

Role of the cytoskeleton in nuclear import.

The role of the cytoskeleton in regulating the intracellular localization of cellular organelles, viruses, and individual proteins has been the subject of much investigation in recent years. While regulated transport through the nuclear pore remains the primary determinant of nuclear localization, it has become clear that the nuclear localization of viruses and some cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation in close proximity to the nuclear pore complex. We also discuss how the size of virions and the viscous nature of the cytoplasm would make it very unlikely or even impossible for viruses to achieve this localization by diffusion alone in the absence of active transport mechanisms. This review focuses on the known methods employed by different viruses and proteins to effect their perinuclear accumulation.

Active Transport, Cell Nucleus↗
PubMed2002

Integrating supports in assessment and planning.

A systematic approach for addressing the support needs of persons with mental retardation and related developmental disabilities is presented and a new scale to measure individual differences in support needs described. The process employed in developing the scale is explained, including the establishment of a typology of support areas that was drawn from a review of the professional literature, a validation process using Q-sort methodology, and a pilot field test. Critical issues and practical challenges associated with efforts to measure and address the support needs of individuals are discussed.

Adult↗