Responses of a Variety of Normal and Malignant Cells to Continuous Cultivation, and Some Practical Applications of These Responses to Problems in the Biology of Disease

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. During the late phase of human adenovirus type 5 (Ad5) infection, cellular protein synthesis is severely inhibited, while viral late proteins are made in large quantities (3, 6). Such preferential expression of viral genes is the result of posttranscriptional regulatory mechanisms: several viral gene products, including VA-RNA1 and the L4 100-kDa protein, contribute to selective translation of viral late mRNAs (see references 18, 64, and 85 for reviews), while the E1B 55-kDa and E4 Orf 6 proteins induce selective export of these mRNAs from the nucleus (reviewed in references 25, 33, and 39). In infected cells, these last two early proteins form a complex (84) that has been implicated in regulation of mRNA export (12, 19). Indeed, the E1B 55-kDa and E4 Orf 6 proteins colocalize to specific sites within infected cell nuclei, the peripheral zones of replication centers (70). Transcription of viral late genes and at least initial processing of viral pre-mRNAs take place at these same sites (4,14,74,75). Mutations that prevent synthesis of the E4 Orf 6 protein or reduce binding of this to the E1B 55-kDa protein (83) result in both mislocalization of the E1B 55-kDa protein and defects in export of viral late mRNAs (39, 70). These properties indicate that E4 Orf 6 protein-dependent recruitment of the E1B protein to the specialized nuclear sites at which viral late pre-mRNAs are synthesized promotes selective export of the processed mRNAs. The observation that the accumulation of viral mRNAs at enlarged interchromatin granules, which form in infected cells as the late phase progresses, correlates with efficient late mRNA export (4, 13) provides further support for the view that efficient mRNA export is intimately coupled to the organization of infected cell nuclei. However, the molecular basis of such coupling remains unknown, nor has the cellular export pathway by which viral late mRNAs are transported from the nucleus to the cytoplasm been identified.AThe E1B 55-kDa protein contains a leucine-rich nuclear export signal (NES) that is recognized by the cellular exportin 1 export receptor and mediates shuttling of the viral protein when it is synthesized either alone or in Ad5-infected cells (26,58). It has also been reported that the E4 Orf 6 protein contains a similar NES nece...

Section: Discussionmentioning

confidence: 99%

Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

González

Wen-ying

Finnen

et al. 2006

“…The method was later applied to research in virology, presumably for directly depositing viral particles on cells to enhance infection (13). The mechanical stress introduced during spinning was also believed to be somewhat beneficial to infection, increasing cell permissiveness (17,18).…”

mentioning

confidence: 99%

Spinoculation Triggers Dynamic Actin and Cofilin Activity That Facilitates HIV-1 Infection of Transformed and Resting CD4 T Cells

Guo

Wang

et al. 2011

Centrifugal inoculation, or spinoculation, is widely used in virology research to enhance viral infection. However, the mechanism remained obscure. Using HIV-1 infection of human T cells as a model, we demonstrate that spinoculation triggers dynamic actin and cofilin activity, probably resulting from cellular responses to centrifugal stress. This actin activity also leads to the upregulation of the HIV-1 receptor and coreceptor, CD4 and CXCR4, enhancing viral binding and entry. We also demonstrate that an actin inhibitor, jasplakinolide, diminishes spin-mediated enhancement. In addition, small interfering RNA (siRNA) knockdown of LIMK1, a cofilin kinase, decreases the enhancement. These results suggest that spin-mediated enhancement cannot be explained simply by a virus-concentrating effect; rather, it is coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, viral entry, and postentry processes. Our results highlight the importance of cofilin and a dynamic cytoskeleton for the initiation of viral infection. Our results also indicate that caution needs to be taken in data interpretation when cells are spinoculated; some of the spin-induced cellular permissiveness may be beyond the natural capacity of an infecting virus.

“…Early on in these studies, we found that our infection efficiencies were substantially enhanced by spinoculation, or centrifugal infection, techniques. Although such protocols have been used in clinical microbiology since the 1950s (18) to enhance infection by a number of difficult-to-culture pathogens (24), including HIV-1 (20,37) and other retroviruses (3,5), the mechanism whereby centrifugation leads to an enhanced level of infection remains controversial (24). Specifically, some groups have proposed that centrifugation enhances cellular susceptibility (22,23) or viral fusion (43), whereas others have suggested that increased viral deposition is the most important aspect of centrifugal enhancement of infection (21,34).…”

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confidence: 99%

Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding

O’Doherty¹,

Swiggard

Malim

2000

624

589

The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1 IIIB virions at 1,200 ؋ g for 2 h at 25°C increases the number of particles that bind to CEM-SS T-cell targets by ϳ40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37°C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, ϳ100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24 gag . Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.