Cystic fibrosis tnsmembrane conductance regulator (CFTR) generates cAMP-regulated channels; mutations in CFFR cause defective Cl-channel function in cystic fibrosis epithelia. We used the patch-chmp technique to determine the single channel properties of Cl-channels in cells expressing recombinant CFIR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl-channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical a-solutions, the channel had a channel conductance of 10.4±0.2 (a = 7) pS and a linear current-voltage relation. The channel was more permeable to Cla than to I-and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of a-channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFIR-generated channels allows further studies oftheir regulation and the mechanism of ion permeation. (J. Cli,. Invest.

Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. A lphaherpesviruses depend upon highly evolved mechanisms to move from mucosal epithelial tissues within neuronal axons to ganglia where latency is established. Following reactivation from latency, virus particles move from ganglia back to peripheral tissues for spread to other hosts. This anterograde transport involves fast axon transport involving microtubules and kinesin motors that propel viral particles from neuron cell bodies (in ganglia) over large distances to axon tips.Depending upon the strain of alphaherpesvirus and the type of neuron, anterograde transport can apparently involve either fully assembled virions or unenveloped capsids (reviewed in references1, 2, and3). Fully assembled, enveloped virions or "Married" particles (4) are produced by capsid envelopment in the cytoplasm of neuron cell bodies, while "Separate" (4) unenveloped capsids (lacking viral glycoproteins) become enveloped at or near axon tips. Early electron microscopy (EM) studies produced evidence for Separate herpes simplex virus (HSV) capsids in human and rat neuronal axons (5-7). Other, more recent EM studies observed a mixture of Separate capsids (25%) and Married particles for two HSV strains (8), but this ratio was reversed, so that 70% of the particles in axons were Separate particles with another HSV strain (T. Mettenleiter, personal communication). Our antibody staining of HSV-infected human neuroblastoma cells produced evidence for mainly Separate capsids and distinct glycoprotein-containing vesicles (4, 9, 10). EM and fluorescent protein analyses of pig pseudorabies virus (PRV) strongly support only Married transport (11)(12)(13)(14). A study involving a "two-color" HSV recombinant expressing a fluorescent glycoprotein and capsids concluded that most HSV anterograde transport involved Married particles (15). Using another "two-color" HSV recombinant expressing fluorescent capsids and glycoproteins gB, we concluded that a majority of capsids moving in rat superior cervical ganglion (SCG) neurons were Separate particles (60%) (16). Thus, we believe that both modes of transport are possible and, in fact, occur.HSV and PRV express two membrane proteins, gE/gI and US9, which are key to the understanding of anterograde transport in neuronal axons (reviewed in references 2 and3). gE/gI is a heterodimer, with both gE and gI required for function, and possesses both substantial extracellular domains and ϳ100-amino-acid (aa) cytoplasmic domains with acidic clusters, ...

Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16–50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

Anterograde transport of herpes simplex virus (HSV) from neuronal cell bodies into, and down, axons is a fundamentally important process for spread to other hosts. Different techniques for imaging HSV in axons have produced two models for how virus particles are transported in axons. In the Separate model, viral nucleocapsids devoid of the viral envelope and membrane glycoproteins are transported in axons. In the Married model, enveloped HSV particles (with the viral glycoproteins) encased within membrane vesicles are transported in the anterograde direction. Earlier studies of HSV-infected human neurons involving electron microscopy (EM) and immunofluorescence staining of glycoproteins and capsids supported the Separate model. However, more-recent live-cell imaging of rat, chicken, and mouse neurons produced evidence supporting the Married model. In a recent EM study, a mixture of Married (75%) and Separate (25%) HSV particles was observed. Here, we studied an HSV recombinant expressing a fluorescent form of the viral glycoprotein gB and a fluorescent capsid protein (VP26), observing that human SK-N-SH neurons contained both Separate (the majority) and Married particles. Live-cell imaging of rat superior cervical ganglion (SCG) neuronal axons in a chamber system (which oriented the axons) also produced evidence of Separate and Married particles. Together, our results suggest that one can observe anterograde transport of both HSV capsids and enveloped virus particles depending on which neurons are cultured and how the neurons are imaged.Herpes simplex virus (HSV) and other alphaherpesviruses establish latency in the sensory nervous system. Periodic reactivation leads to the production of infectious virus in sensory ganglia, followed by virus transport in neuronal axons to epithelial tissues. Repetitive infection of the cornea causes scarring, which represents the major infectious cause of blindness. Anterograde transport (from neuronal cell bodies to axon termini) is a fundamentally important property of alphaherpesviruses, essential for long-term survival in the form of spread to other hosts. Early studies of HSV infection in human fetal neurons involving electron microscopy (EM), immuno-EM, and immunofluorescence analyses led to the conclusion that capsids are transported in the anterograde direction separately from vesicles containing viral glycoproteins (9, 17). More-recent studies from the same laboratory showed that there was virus envelopment (assembly of capsids with glycoproteins) at relatively numerous varicosities and at growth cones in cultured human neurons (18). Studies in our laboratory also supported what we termed the "Separate" model for HSV anterograde transport, i.e., transport of unenveloped capsids separately from viral glycoproteins (20)(21)(22). In this model, envelopment occurs at axon termini. In human neuroblastoma (SK-N-SH) cells differentiated to produce neurites, HSV glycoproteins stained with a panel of different antibodies (against gB, gD, gE, or gI) were observed as puncta that w...

Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE US9 double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE US9 mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde transport) or from cell bodies to axon tips (anterograde transport). This transit in axons is essential for alphaherpesviruses to establish latency in ganglia and then to reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins: gE/gI and US9. Our studies reveal new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm, which blocks anterograde transport of enveloped particles. In addition, there are defects in the sorting of virus particles such that particles, when formed, do not enter proximal axons.