Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus.

To study the function of the envelope glycoprotein gH of pseudorabies virus, a gH null mutant was constructed. A premature translation termination codon was introduced in the gH gene by linker insertion mutagenesis, and a mutant virus was rescued by using a cell line that expresses the wild-type protein. Mutant virus isolated from complementing cells was unable to form plaques on noncomplementing cells, indicating that gH is essential in the life cycle of the virus. Immunological staining and electron microscopy showed that the mutant virus produced noninfectious progeny and was unable to spread from infected to uninfected cells by cell-cell fusion. Thus, similar to gH of herpes simplex virus, gH of pseudorabies virus is required for entry and cell-to-cell spread.     

Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.

Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is not required for the subsequent steps in virus replication. Phenotypically complemented gD mutants can infect cells and can spread, both in vitro and in mice, by direct cell-to-cell transmission. Progeny virions released by infected cells are noninfectious because they lack gD. The aim of this study was to determine the role of gD in the neuropathogenicity of PRV in its natural host, the pig. We investigated whether gD-negative PRV can spread transneuronally via synaptically linked neurons of the olfactory and trigeminal routes. High doses of a phenotypically complemented gD mutant and gD mutants that are unable to express either gI or gI plus gE were inoculated intranasally in 3- to 5-week-old pigs. Compared with the wild-type virus, the virulence of the gD mutant was reduced. However, pigs inoculated with the gD mutant still developed fever and respiratory signs. Additional inactivation of either gI or gI plus gE further decreased virulence for pigs. Immunohistochemical examination of infected pigs showed that a PRV gD mutant could replicate and spread transneuronally into the central nervous system (CNS). Compared with the wild-type virus, the gD mutant had infected fewer neurons of the CNS on day 2. Nevertheless, on day 3, the gD-negative PRV had infected more neurons and viral antigens were present in second- and third-order neurons in the olfactory bulb, brain stem, and medulla oblongata. In contrast, gD mutants which are unable to express either gI or gI plus gE infected a limited number of first-order neurons in the olfactory epithelium and in the trigeminal ganglion and did not spread transneuronally or infect the CNS. Thus, transsynaptic spread of PRV in pigs can occur independently of gD. Possible mechanisms of transsynaptic transport of PRV are discussed.     

Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.     

Complementation analysis of pseudorabies virus gE and gI mutants in retinal ganglion cell neurotropism.

Pseudorabies virus glycoproteins gE and gI are required to infect some, but not all, regions of the rodent central nervous system after peripheral injection. After infection of the retina, pseudorabies virus mutants lacking either gE or gI can subsequently infect neural centers involved in the control of circadian function but cannot infect visual circuits mediating visual perception or the reflex movement of the eyes. In this study, we used genetic complementation to test the hypothesis that gE and gI are required for entry into the specific retinal ganglion cells that project to visual centers. These data strongly suggest that gE and gI must function after the viruses enter primary neurons in the retina.     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

Role of pseudorabies virus glycoprotein gI in virus release from infected cells.

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.     

Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein

Envelope glycoproteins gH and gL, which form a complex, are conserved throughout the family Herpesviridae. The gH-gL complex is essential for the fusion between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several herpesviruses that gL is required for proper folding, intracellular transport, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-DeltagLbeta, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, resulting in only single or small foci of infected cells (B. G. Klupp, W. Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687-7695, 1997). We used this limited cell-to-cell spreading ability of PrV-DeltagLbeta for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate, designated PrV-DeltagLPass, was further characterized. To identify the mutation leading to this gL-independent infectious phenotype, Southern and Western blot analyses, radioimmunoprecipitations, and DNA sequencing were performed. The results showed that rearrangement of a genomic region comprising part of the gH gene into a duplicated copy of part of the unique short region resulted in a fusion fragment predicted to encode a protein consisting of the N-terminal 271 amino acids of gD fused to the C-terminal 590 residues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-specific antibodies verified the presence of a gDH fusion protein. To prove that this fusion protein mediates infectivity of PrV-DeltagLPass, cotransfection of PrV-DeltagLbeta DNA with the cloned fusion fragment was performed, and a cell line, Nde-67, carrying the fusion gene was established. After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-DeltagLbeta on Nde-67 cells produced infectious virions. Thus, a gDH fusion polypeptide can compensate for function of the essential gL in entry and cell-to-cell spread of PrV.     

Role of cell-associated enveloped vaccinia virus in cell-to-cell spread.

The roles of intracellular naked (INV), cell-associated enveloped (CEV), and extracellular enveloped (EEV) forms of vaccinia virus in cell-to-cell and longer-range spread were investigated by using two closely related strains of vaccinia virus, WR and IHD-J. We confirmed previous results that WR and IHD-J produced similar amounts of INV and formed similar-size primary plaques but that IHD-J produced 10 to 40 times more EEV and spread to distant cells much more efficiently than did WR. Nevertheless, cells infected with WR and IHD-J had similar amounts of CEV, indicating that wrapping and transport of WR virions were unimpaired. A WR mutant with a deletion in VP37, the major outer envelope protein, formed normal amounts of INV; however, the generation of CEV was blocked and plaque formation was inhibited. These results suggested that CEV is the form of virus that mediates cell-to-cell spread. Marker rescue experiments indicated that the differences in EEV production by WR and IHD-J were not due to sequence differences in VP37. The low amount of WR EEV could be attributed to retention of CEV on the cell membrane. In support of this hypothesis, mild treatment with trypsin released as much or more infectious virus from cells infected with WR as it did with cells infected with IHD-J. Most of the virus released by trypsin sedimented with the buoyant density of EEV. Also, addition of trypsin to cells following inoculation with WR led to a comet-shaped distribution of secondary plaques characteristic of IHD-J. These results demonstrated that the release of CEV from the cell surface was limiting for extracellular virus formation and affirmed the role of EEV in long-range spread.     

Complex between glycoproteins gI and gp63 of pseudorabies virus: its effect on virus replication.

To ascertain the biological functions of different glycoproteins that are nonessential for pseudorabies virus growth in vitro, we have constructed mutants defective in one (or a combination) of these glycoproteins and have examined various aspects of their role in the infective process. We made the following two observations. (i) Glycoproteins gI and gp63 are noncovalently complexed to each other. They are coprecipitated by antisera against either one of these glycoproteins but do not share antigenic determinants: monoclonal antibodies against gp63 do not immunoprecipitate gI from extracts of gp63- mutant-infected cells, and monoclonal antibodies against gI do not immunoprecipitate gp63 from extracts of gI- mutant-infected cells. (ii) Mutants unable to synthesize either gI or gp63 have some common biological characteristics; they have a growth advantage in primary chicken embryo fibroblasts. Furthermore, we have shown previously that in conjunction with glycoprotein gIII, gI and gp63 are necessary for the expression of virulence (T. C. Mettenleiter, C. Schreurs, F. Zuckermann, T. Ben-Porat, and A. S. Kaplan, J. Virol. 62, 2712-2717, 1988). These results show that the functional entity affecting virus replication in chicken embryo fibroblasts, as well as affecting virulence, is the complex between gI and gp63. The gI-gp63 complex of pseudorabies virus does not appear to have Fc receptor activity as does its homolog, the gI-gE complex of herpes simplex virus.     

An important role for syndecan-1 in herpes simplex virus type-1 induced cell-to-cell fusion and virus spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.     

Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system

The molecular mechanisms underlying the directional neuron-to-epithelial cell transport of herpesvirus particles during infection are poorly understood. To study the role of the viral glycoprotein D (gD) in the directional spread of herpes simplex virus (HSV) and pseudorabies virus (PRV) infection, a culture system consisting of sympathetic neurons or epithelial cells in different compartments was employed. We discovered that PRV infection could spread efficiently from neurons to cells and back to neurons in the absence of gD, the viral ligand required for entry of extracellular particles. Unexpectedly, PRV infection can also spread transneuronally via axo-axonal contacts. We show that this form of interaxonal spread between neurons is gD independent and is not mediated by extracellular virions. We also found that unlike PRV gD, HSV-1 gD is required for neuron-to-cell spread of infection. Neither of the host cell gD receptors (HVEM and nectin-1) is required in target primary fibroblasts for neuron-to-cell spread of HSV-1 or PRV infection.     

The extracellular domain of herpes simplex virus gE is indispensable for efficient cell-to-cell spread: evidence for gE/gI receptors

Herpes simplex virus (HSV) spreads rapidly and efficiently within epithelial and neuronal tissues. The HSV glycoprotein heterodimer gE/gI plays a critical role in promoting cell-to-cell spread but does not obviously function during entry of extracellular virus into cells. Thus, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. There was previous evidence that the large extracellular (ET) domains of gE/gI might be important in cell-to-cell spread. First, gE/gI extensively accumulates at cell junctions, consistent with being tethered there. Second, expression of gE/gI in trans interfered with HSV spread between epithelial cells. To directly test whether the gE ET domain was necessary for gE/gI to promote virus spread, a panel of gE mutants with small insertions in the ET domain was constructed. Cell-to-cell spread was reduced when insertions were made within either of two regions, residues 256 to 291 or 348 to 380. There was a strong correlation between loss of cell-to-cell spread function and binding of immunoglobulin. gE ET domain mutants 277, 291, and 348 bound gI, produced mature forms of gE that reached the cell surface, and were incorporated into virions yet produced plaques similar to gE null mutants. Moreover, all three mutants were highly restricted in spread within the corneal epithelium, in the case of mutant 277 to only 4 to 6% of the number of cells compared with wild-type HSV. Therefore, the ET domain of gE is indispensable for efficient cell-to-cell spread. These observations are consistent with our working hypothesis that gE/gI can bind extracellular ligands, so-called gE/gI receptors that are concentrated at epithelial cell junctions. This fits with similarities in structure and function of gE/gI and gD, which is a receptor binding protein.     

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.     

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.     

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.     

Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function.     

HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores

Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.     

Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus.

We previously observed that when human immunodeficiency virus (HIV)-infected T lymphocytes are added to epithelial cells, they adhere, polarize, and secrete virions unidirectionally onto the epithelium. Epithelial cells subsequently take up virus and become productively infected. We report here that colchicine treatment of T-lymphocyte suspensions induced lymphocyte polarization, redistribution of F-actin into a pseudopod, and secretion of HIV from the pseudopod. Immobilization of T lymphocytes on negatively charged plastic also caused redistribution of F-actin and unidirectional secretion of HIV onto the plastic. As neither colchicine nor adhesion caused an increase in HIV secretion, they apparently act by focusing secretion to the tip of the pseudopod. We speculate that adhesion-induced polar secretion of HIV, from activated mononuclear cells onto epithelia, is a cytoskeleton-mediated process which may be involved in HIV transmission in vivo.     

Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment,penetration, growth curve and direct cell-to-cell spread

The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.