The cellular immune response to primary influenza virus infection is complex, involving multiple cell types and anatomical compartments, and is difficult to measure directly. Here we develop a two-compartment model that quantifies the interplay between viral replication and adaptive immunity. The fidelity of the model is demonstrated by accurately confirming the role of CD4 help for antibody persistence and the consequences of immune depletion experiments. The model predicts that drugs to limit viral infection and/or production must be administered within 2 days of infection, with a benefit of combination therapy when administered early, and cytotoxic CD8 T cells in the lung are as effective for viral clearance as neutralizing antibodies when present at the time of challenge. The model can be used to investigate explicit biological scenarios and generate experimentally testable hypotheses. For example, when the adaptive response depends on cellular immune cell priming, regulation of antigen presentation has greater influence on the kinetics of viral clearance than the efficiency of virus neutralization or cellular cytotoxicity. These findings suggest that the modulation of antigen presentation or the number of lung resident cytotoxic cells and the combination drug intervention are strategies to combat highly virulent influenza viruses. We further compared alternative model structures, for example, B-cell activation directly by the virus versus that through professional antigen-presenting cells or dendritic cell licensing of CD8 T cells.

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr 112 and Tyr 132 of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 m/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.The infectious forms of some enveloped viruses are assembled at the plasma membrane, providing direct access to the extracellular environment. Other viruses, however, are assembled in the nucleus or internal regions of the cytoplasm and require locomotion to reach the periphery of the cell. Such movement is likely to involve the actin or microtubule cytoskeleton (28). Infectious intracellular mature vaccinia virus virions (IMV) form in juxtanuclear factory regions of the cytoplasm (5, 19) and are wrapped by a double membrane derived from trans-Golgi or endosomal cisternae to become intracellular enveloped virions (IEV), which are then translocated to the periphery of the cell where the outer IEV and plasma membranes fuse (12,15,19,26,31). The plasma membrane adherent and the released extracellular virions are called cell-associated enveloped virions (CEV) and extracellular enveloped virions (EEV), respectively. The association of actin filaments with CEV at the tips of specialized microvilli has been appreciated since the early electron and immunofluorescence microscopic studies of Stokes (29) and Hiller e...

We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 m/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.Vaccinia virus morphogenesis is a complex process that begins with the formation of crescent membranes within cytoplasmic factory regions and leads to the production of infectious intracellular mature virions (IMV) (6,13,19,38). After IMV are transported away from the factories, some are wrapped with a double membrane derived from the transGolgi network (TGN) or endosomal cisternae to form intracellular enveloped virions (IEV) (15,36,40). By associating with actin tails (4) or through other mechanisms (41, 44), the IEV reach the periphery of the cell, where one of the two outer membranes is thought to fuse with the plasma membrane. The externalized virions remain attached to the outer surface of the cell as cell-associated extracellular enveloped virions or are released as extracellular enveloped virions (EEV). The cellassociated extracellular enveloped virions and EEV are thought to be responsible for cell-to-cell (2) and long-range (26) virus spread, respectively.The proteins encoded by the F13L, B5R, A33R, A34R, A36R, and A56R open reading frames (ORFs) are constituents of the IEV or EEV membrane (7,9,20,25,28,32,41). Deletion of any one of these ORFs except A56R, which encodes the viral hemagglutinin, resulted in a mutant virus with a small-plaque phenotype. The F13L and B5R proteins are required for EEV formation, because del...