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Vaccinia virus cores are transported on microtubules
Gemma C Carter1, Gaener Rodger2,1, Brendan J Murphy1
1Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Abstract:
Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.
Insights
Vaccinia virus (VV) cores use microtubules for intracellular transport after entry. This movement is essential for infection and is inhibited by microtubule-disrupting drugs, but not actin inhibitors.
Area of Science:
- Virology
- Cell Biology
- Microbiology
Background:
- Vaccinia virus (VV) exists in multiple forms, including intracellular mature virus (IMV).
- Understanding the intracellular transport of incoming VV cores is crucial for deciphering viral infection mechanisms.
Purpose of the Study:
- To investigate the intracellular transport pathway of incoming VV cores derived from IMV.
- To determine the role of cellular structures, such as microtubules and actin filaments, in VV core intracellular movement.
Main Methods:
- Generation of recombinant VVs expressing enhanced green fluorescent protein (EGFP)-fused A5L virus core protein.
- Immunoblotting and immunoelectron microscopy to confirm fusion protein incorporation into VV cores.
- Confocal microscopy to track the movement of fluorescently labeled VV cores in infected cells.
- Treatment of infected cells with nocodazole, colchicine, or cytochalasin D to assess the involvement of microtubules and actin.
Main Results:
- Recombinant VVs expressing A5L-EGFP fusion proteins were viable and incorporated into VV cores.
- Intracellular VV cores co-localized with microtubules and exhibited stop-start movement.
- The average speed of core movement was measured at 51.8 µm/min.
- Microtubule-disrupting agents (nocodazole, colchicine) inhibited core movement, while cytochalasin D did not.
Conclusions:
- VV cores derived from IMV utilize microtubules for their intracellular transport after cell entry.
- This microtubule-dependent transport is a key step in the early stages of VV infection.