HIV-1 subverts antigen processing in dendritic cells (DCs) resulting in viral uptake, infection, and transfer to T cells. Although DCs bound monomeric gp120 and HIV-1 similarly, virus rarely colocalized with endolysosomal markers, unlike gp120, suggesting HIV-1 alters endolysosomal trafficking. Virus within DC intracellular compartments rapidly moved to DC-CD4 ؉ lymphocyte synapses when introduced to CD4 ؉ lymphocyte cultures.Although viral harboring and transfer from nonlysosomal compartments was transient, given DC-associated virus protein, nucleic acids, and infectious HIV-1 transfer to CD4 ؉ , lymphocytes decayed within 24 hours. However a second long-term transfer phase was apparent in immature DCs after 48 hours as a zidovudinesensitive rise in proviral DNA. Therefore, DCs transfer HIV-1 to CD4 ؉ lymphocytes in 2 distinct phases. Immature and mature DCs first divert virus from the endolysosomal pathway to the DC-T-cell synapse. Secondly, the later transfer phase from immature DCs is through de novo HIV-1 production. Thus, the controversy of DCs being infected or not infected for the mechanics of viral transfer to CD4 ؉ lymphocytes can be addressed as a function of time.

The mechanism of anterograde transport of herpes simplex virus was studied in cultured dissociated human and rat dorsal root ganglion neurons. The neurons were infected with HSV-1 to examine the distribution of capsid (VP5), tegument (VP16), and glycoproteins (gC and gB) at 2, 6, 10, 13, 17, and 24 h postinfection (p.i.) with or without nocodazole (a microtubule depolymerizer) or brefeldin A (a Golgi inhibitor). Retrogradely transported VP5 was detected in the cytoplasm of the cell body up to the nuclear membrane at 2 h p.i. It was first detected de novo in the nucleus and cytoplasm at 10 h p.i., the axon hillock at 13 h p.i., and the axon at 15 to 17 h p.i. gC and gB were first detected de novo in the cytoplasm and the axon hillock at 10 h p.i. and then in the axon at 13 h p.i., which was always earlier than the detection of VP5. De novo-synthesized VP16 was first detected in the cytoplasm at 10 to 13 h p.i. and in the axon at 16 to 17 h p.i. Nocodazole inhibited the transport of all antigens, VP5, VP16, and gC or gB. The kinetics of inhibition of VP5 and gC could be dissociated. Brefeldin A inhibited the transport of gC or gB and VP16 but not VP5 into axons. Transmission immunoelectron microscopy confirmed that there were unenveloped nucleocapsids in the axon with or without brefeldin A. These findings demonstrate that glycoproteins and capsids, associated with tegument proteins, are transported by different pathways with slightly differing kinetics from the nucleus to the axon. Furthermore, axonal anterograde transport of the nucleocapsid can proceed despite the loss of most VP16.HSV-1 enters the human body via the mucosa or skin and then the termini of neurons within the epidermis and is retrogradely transported to the cell bodies of neurons in the DRG, where it becomes latent. Reactivation of HSV-1 from latency during a patient's lifetime is very frequent, resulting in symptomatic disease or, more commonly, unrecognized lesions and asymptomatic shedding (10, 43). Latency and other stages of the viral infection cycle have been well studied in experimental animals in vivo. Retrograde transport of HSV in rat DRG neurons was demonstrated to be microtubule associated, and virions travel as unenveloped nucleocapsids (19,23). However, the events following reactivation have not been well characterized. The development of a model of interaction between the outgrowing axons of human fetal DRG and epidermal explants in separate chambers of a two-chamber in vitro system in our laboratory allowed studies of the transport of HSV-1 from the cell bodies of DRG neurons along the principal axon to epidermal cells (17,31,32). The rate of anterograde transport of nucleocapsids and glycoproteins was estimated by immunofluorescence and confocal microscopy at 0.6 mm per s, consistent with rapid microtubule-associated transport (28). TEM of cross sections of axons behind the advancing front of viral antigen showed that only unenveloped nucleocapsids adjacent to microtubules were present. Recent studies using scanning immunoelectro...

HSV efficiently infects dendritic cells (DCs) in their immature state and induces down-regulation of costimulatory and adhesion molecules. As in mice, HSV infection of human DCs also leads to their rapid and progressive apoptosis, and we show that both early and late viral proteins contribute to its induction. Because topical HSV infection is confined to the epidermis, Langerhans cells are expected to be the major APCs in draining lymph nodes. However, recent observations in murine models show T cell activation to be mediated by nonepidermal DC subsets, suggesting cross-presentation of viral Ag. In this study we provide an explanation for this phenomenon, demonstrating that HSV-infected apoptotic DCs are readily phagocytosed by uninfected bystander DCs, which, in turn, stimulate virus-specific CD8+ T cell clones.

The mechanisms of axonal transport of the alphaherpesviruses, HSV and pseudorabies virus (PrV), in neuronal axons are of fundamental interest, particularly in comparison with other viruses, and offer potential sites for antiviral intervention or development of gene therapy vectors. These herpesviruses are transported rapidly along microtubules (MTs) in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterogradely in the opposite direction. Retrograde transport follows fusion and deenvelopment of the viral capsid at the axonal membrane followed by loss of most of the tegument proteins and then binding of the capsid via one or more viral proteins (VPs) to the retrograde molecular motor dynein. The HSV capsid protein pUL35 has been shown to bind to the dynein light chain Tctex1 but is likely to be accompanied by additional dynein binding of an inner tegument protein. The mechanism of anterograde transport is much more controversial with different processes being claimed for PrV and HSV: separate transport of HSV capsid/tegument and glycoproteins versus PrV transport as an enveloped virion. The controversy has not been resolved despite application, in several laboratories, of confocal microscopy (CFM), realtime fluorescence with viruses dual labelled on capsid and glycoprotein, electron microscopy in situ and immunoelectron microscopy. Different processes for each virus seem counterintuitive although they are the most divergent in the alphaherpesvirus subfamily. Current hypotheses suggest that unenveloped HSV capsids complete assembly in the axonal growth cones and varicosities, whereas with PrV unenveloped capsids are only found travelling in a retrograde direction.

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26؉ capsids bound to RP3; VP26؊ capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37°C. After 1 h VP26؉ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26؉ capsids had moved closer to the cell nucleus, whereas VP26؊ capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.

After infection of skin or mucosa, herpes simplex virus enters the sensory nerve endings and is conveyed by retrograde axonal transport to the dorsal root ganglion, where the virus develops lifelong latency. Intermittent reactivation, which is spontaneous in humans, leads to anterograde transport of virus particles and proteins to the skin or mucosa, where the virus is shed and/or causes disease. Immune control of viral infection and replication occurs at the level of skin or mucosa during initial or recurrent infection and also within the dorsal root ganglion, where immune mechanisms control latency and reactivation. This article examines current views on the mechanisms of retrograde and anterograde transport of the virus in axons and the mechanisms of innate and adaptive immunity that control infection in the skin or mucosa and in the dorsal root ganglion--in particular, the role of interferons, myeloid and plasmacytoid dendritic cells, CD4(+) and CD8(+) T cells, and interferon- gamma and other cytokines, including their significance in the development of vaccines for genital herpes.

Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons.

Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h postinfection, wild-type (wt) HSV-2 (186) abortively infected murine bone marrow-derived DC and induced early cell death compared to UV-inactivated HSV-2 or mock-infected DC. HSV-2-induced loss of DC viability was more rapid than that induced by HSV-1 and was due, in part, to apoptosis, as shown by TEM, caspase-3 activation, and terminal deoxynucleotidyl transferase-mediated dCTP biotin nick end labeling. HSV induced type-specific changes in the murine DC immunophenotype. At 12 h postinfection, wt HSV-2 upregulated DC major histocompatibility complex (MHC) class II expression, and in contrast to UV-inactivated HSV-2, downregulated expression of MHC class I, but it had no effect on surface CD40, CD80, or CD86. Wt HSV-1 (MC-1) induced only CD40 upregulation. More-profound effects on the DC immunophenotype were observed in HSV-2-infected neonatal DC. Wt HSV of either serotype impaired murine DC-induced T-cell alloproliferation and lipopolysaccharideinduced DC interleukin-12 secretion. Thus, there are marked differences in the levels of HSV-induced cytolysis in DC according to the HSV serotype, although HSV-2 displays immunomodulatory effects on the DC immunophenotype and function similar to those of HSV-1.Dendritic cells (DC) play a key role in the induction of the primary cellular immune response to intracellular pathogens like herpes simplex virus (HSV), as they are the main cell type that stimulates naive T cells in the draining lymph nodes (23). The strength and character of the antigen-specific T-cell response are determined by factors such as the level of costimulatory molecule (B-7) expression and the density of antigen expressed on the surfaces of DC (1, 33). DC are also thought to be the major source of interleukin-12 (IL-12) (31), which has been demonstrated to play a pivotal role in the differentiation of naive CD4 ϩ T cells into type 1 T helper (Th1) cells (18), although recent studies suggest that IL-12 produced by DC is required for optimal T-cell gamma interferon production rather than for CD4 ϩ -T-cell polarization (26). HSV type 1 (HSV-1) infection of adult human DC derived from peripheral blood monocytes (MoDC) has been shown by a number of groups (15,19,27) to impair costimulatory molecule upregulation of immature DC. However, the timing and degree of this impairment and the presence or absence of associated effects on major histocompatibility complex (MHC) class I (MHC-I) and MHC-II molecule expression were different, possibly due to differences in the types of HSV-1 strain used. Infection of immature MoDC with a disabled infectious single-cycle mutant (DISC-HSV-1-GFP) inhibited DC maturation (as shown by downregulation of costimulatory molecules) and induced marked downregulation of MHC-I expression, attributed...