Matrix Fibronectin Binds Gammaretrovirus and Assists in Entry: New Light on Viral Infections

Abstract

A major entry route for the gammaretrovirus amphotropic murine leukemia virus (A-MLV) into NIH 3T3 fibroblasts is via caveola-dependent endocytosis. However, during the infection time, few viral particles can be observed intracellularly. Analyzing the dynamics of the A-MLV infection process by using total internal reflection fluorescence microscopy, we show that the majority of viruses are extracellular and bound to the fibronectin matrix. Moreover, the amounts of bound virus and of fibronectin correlated. Using confocal microscopy, nanoparticles targeted to fibronectin by a III1C-fibronectin fragment or anti-fibronectin antibody were detected intracellularly in NIH 3T3 cells; unconjugated nanoparticles neither bound to cells nor were detectable intracellularly. Furthermore, A-MLV colocalized intracellularly with the fibronectin-targeted nanoparticles, suggesting that they were taken up by the same cellular pathway. Both A-MLV entry and fibronectin turnover depend on caveolar endocytosis, and we found that inhibiting viral binding to the extracellular NIH 3T3 fibronectin-matrix dramatically reduced A-MLV infection, indeed, showing an active role of fibronectin in infection. We suggest that binding to the cellular fibronectin matrix provides a new mechanism by which viruses can enter cells.Knowledge of the role of cellular factors in retroviral entry increases our understanding of how viruses can exploit cellular features to enter host cells. Gammaretroviruses, like other enveloped viruses, are dependent on specific cellular receptors for fusion of the viral membrane with the cellular membrane; e.g., amphotropic murine leukemia virus (A-MLV) depends on the presence of the ubiquitously expressed sodium-dependent phosphate transporter Pit2 (17,29,48,50). MLV-based retroviral vectors including vectors carrying A-MLV envelope proteins are widely used in gene therapy protocols, and although retroviruses and retroviral vectors can infect a variety of dividing cell types, the efficiencies vary greatly among different cell types despite the fact that these cells all express Pit2 (48). Specifically, efficient transduction of hematopoietic cells can only be achieved when infection occurs in the presence of chymotryptic fibronectin (FN) fragments like 30/35 FN, recombinant chimeric FN fragments like CH-296 (RetroNectin) (13, 32), or shed FN (sFN) derived from NIH 3T3-based packaging cell lines (21 and C. S. Søndergaard, C. Haldrup, C. Beer, D. B. Kohn, and L. Pedersen, submitted for publication). It has been suggested that increased infection is due to concomitant binding of vectors and cells to the fibronectin fragments and that this increases the likelihood that a vector and a cell will interact compared to the situation where both vectors and cells would be in suspension (13, 31). In agreement with this hypothesis, we could recently show that gammaretroviral vectors bind to sFN from NIH 3T3 cultures (Søndergaard et al., submitted).However, the role of naturally occurring FN in viral entry is largely unknown.FN is a compo...

Clinical Application of a Retroviral Gene Transfer Protocol Based on Centrifugation-Mediated Vector Preloading

“…Hughes and coworkers (2001) were able to show that retroviral vectors produced by packaging cell lines derived from NIH3T3 were almost completely bound to fibronectin. More recently, a direct supportive effect of NIH3T3-released fibronectin on infectivity of gammaretroviral particles was demonstrated (Beer and Pedersen, 2007). These data are in agreement with our observation that preloading is much less efficient when the supernatant is derived from other, non-NIH3T3-based producer cell lines (e.g., 293T) (B. Fehse and K. Kühlcke, unpublished observations).…”

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

“…Culture medium containing anti-FN or control antibodies was added to the cells at the indicated time points. The concentration tested in the present study was based on other studies (6,20,23). At a concentration of 2 g/ml, the anti-FN antibody level is estimated to be about 7.5Eϩ06 molecules per cell.…”

Cooperative Binding and Activation of Fibronectin by a Bacterial Surface Protein

“…Caveolae have also been implicated in caveolin-1-dependent ␣5␤1 integrin-mediated FN endocytosis (42). These findings suggest the interesting possibility that SfbI-expressing streptococci and potentially other bacterial pathogens or viruses (43), through multiple binding to FN, target this cellular recycling mechanism to achieve internalization. …”