Microtubule-dependent Transport Of Bovine Immunodeficiency Virus

Bovine immunodeficiency virus (BIV) belongs to the lentivirus subfamily of retroviruses, which shares high morphologic and genomic similarity to human immunodeficiency virus (HIV). BIV, which is not oncogenic, results in a wasting syndrome associated with central nervous system lesions and leukosis in infected cattle, and induces the formation of syncytia in cell cultures. Like most lentiviruses, the RNA genome of BIV is reverse-transcribed into DNA and integrated into the host chromosome as provirus.As obligate cell parasites, viruses embezzle the cellular machinery for diverse activities. At early stage of infection, viruses must move from the peripheral region to the perinuclear region to replicate. To hijack the motor proteins and travel along the microtubule network is a universal mechanism for viral transport. As cellular roadway. microtubules are long, hollow cylinders assembled from equally oriented heterodimers ofα-andβ-tubulin, which gives microtubules a structural polarity. In unpolarized cell types, the minus-end is localized at microtubule-organizing center and plus-end is directed towards plasma membrane. Minus-end-directed motion, a process known as retrograde transport, refers to viral movement from the cell periphery to the perinuclear region and is mediated by cytoplasmic dynein. In contrast, plus-end-directed motion, a process known as anterograde transport, refers to viral movement from the assembly site to the cell periphery and mostly requires kinesin family motors. Interaction of viral components with motor protein subunits is crucial for viral trafficking.Cytoplasmic transport of BIV is largely unknown, as well as the molecular machinery of HIV-1 transport. In this study, we first analyzed the effect of microtubule-disrupting agents on BIV infection by using BIV indicator cells. BIV infection was severely inhibited by nocodazole in a dose-dependent manner, particularly at the early infection stage. Microtubule fractionation experiments and examination of acetylated tubulin levels showed that BIV infection promoted microtubule polymerization and increased microtubule stability. To further confirm if microtubules play a role in BIV transport, an immunofluorescent assay was performed to examine the subcellular distribution of viral particles at different hours post infection (hpi). Viral particles were mostly localized at the peripheral region at 2 hpi and moved to the perinuclear region at 8 hpi. In contrast, a majority of viral particles remained at the peripheral region in nocodazole-treated cells at 8 hpi. Quantitative analysis of viral particle distribution showed that nocodazole largely decreased the amount of the perinuclear particles. In addition, immunofluorescent microcopy and electron microscopy showed that the transporting viral particles were localized on the microtubules. These results demonstrate the dependence of microtubules in BIV retrograde transport.Overexpression of Dynamitin disrupts the function of the microtubule-associated motor protein dynein, and inhibited BIV infection in our experiments. Further immunofluorescent microcopy showed that BIV retrograde transport was interrupted by overexpression of Dynamitin, demonstrating that BIV retrograde transport is mediated by dynein motors. By a yeast two-hybrid assay, we found that the capsid protein (CA) of BIV interacted with the dynein light chain component LC8. GST-pulldown, CBP-pulldown and coimmunoprecipitation assays further demonstrated an interaction between CA and LC8 in mammalian cells, in vitro and in the context of infection. LC8-binding sequence in CA was localized at the N-terminal domain of CA protein.Small-hairpin RNAs were used to knockdown LC8 expression. BIV infectivity were largely decreased in LC8-depleted cells, in which BIV retrograde transport was interrupted, as evidenced in an immunofluorescent assay. Examination of microtubule fraction and cytosolic fraction showed that CA was dominantly found in the microtubule fraction in untreated cells. In contrast, shRNAs induced relocation of CA to the cytosol in LC8-depleted cells. Further coimmunoprecipitation assays demonstrated that CA was associated with dynein complex through an interaction with LC8. In agreement with this result, we observed the colocalization of transporting viral particles and GFP-labeled dynein complex by immunofluorescent microsopy. Furthermore, overexpression of CA inhibited BIV infection, raising the possibility that excessive CA could sequester LC8 which is essential for the viral retrograde transport. These data demonstrate that LC8 acts as a critical linker between viral particles and microtubules.Our findings present the first evidence that incoming BIV particles employ host microtubule/dynein machinery for transport towards the perinuclear region. The molecular basis of BIV retrograde transport enriches the knowledge of viral life cycle, and the understanding of the related steps in HIV and other retroviruses. The ultimate goal of lentiviral studies lies in elimination of AIDS. Our data related to viral retrograde transport enable us to block viral infection before integration and develop a potential target for the design of antiviral strategies.