Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

Abstract

During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCERetroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm-a crowded environment where diffusion is slow-is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

Functional Evidence of the Involvement of the Dynein Light Chain DYNLRB2 in Murine Leukemia Virus Infection

“…Several screens have revealed that cytoskeleton-associated proteins are important in different stages of retrovirus replication (8,17). Following that line, we identified dynein and its regulators as important parts of the host cell machinery required for MLV infection (16), with the dynein regulators seeming to be essential for a step before nuclear entry but after reverse transcription. We also reported that the dynein intermediate chain is important for MLV infection, since its knockdown significantly reduced MLV infection.…”

“…We recently demonstrated that the MLV preintegration complex (PIC) associates with dynein and dynein regulators (16); specifically, the dynein intermediate chain DYNC1I2 is required for MLV infection. The dynein intermediate chain is not only important for cargo binding but also essential for the association of dynein light chains with the complex (22).…”

“…We previously showed that dynactin components have a differential effect on the different tropisms of MLV: ecotropic MLV strongly requires them, while amphotropic MLV only mildly requires them (16). Therefore, we tested if infections by amphotropic and vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped MLV strains were or were not reduced in DYNLRB2 KD cells.…”

“…The cytoplasm is extremely crowded, and simple diffusion is predicted to be too slow to explain the kinetics of virus infection (1). Many retroviruses are actively transported along microtubules (2-4) to move through the cytoplasm and reach the nucleus, and the microtubule retrograde transport complex dynein has been shown to have an important role in trafficking and infection for several viral families (2,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).Murine leukemia virus (MLV) belongs to the Gammaretrovirus genus, whose members are strongly dependent on cell division to gain entry into the nucleus. These viruses do not have the ability to cross the nuclear membrane of nondividing cells and rather must wait for nuclear membrane breakdown to occur during mitosis to access host chromatin.…”

“…The cytoplasm is extremely crowded, and simple diffusion is predicted to be too slow to explain the kinetics of virus infection (1). Many retroviruses are actively transported along microtubules (2)(3)(4) to move through the cytoplasm and reach the nucleus, and the microtubule retrograde transport complex dynein has been shown to have an important role in trafficking and infection for several viral families (2,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”