Background Occasional cases of viral escape in cerebrospinal fluid (CSF) despite suppression of plasma HIV-1 RNA have been reported. We investigated CSF viral escape in subjects treated with commonly used ART regimens in relation to intrathecal immune activation and CNS penetration effectiveness (CPE) rank. Methods Sixty-nine neurologically asymptomatic subjects treated with ART >6 months and plasma HIV-1 RNA <50 copies/ml were cross-sectionally included in the analysis. ART regimens included efavirenz, lopinavir/ritonavir or atazanavir/ritonavir combined with tenofovir, abacavir or zidovudine and emtricitabine or lamivudine. HIV-1 RNA was analysed with real-time PCR assays. Neopterin was analysed by ELISA. Results Seven (10%) of the 69 subjects had detectable CSF HIV-1 RNA, in median (IQR) 121 (54–213) copies/ml. Subjects with detectable CSF virus had significantly higher CSF neopterin and longer duration of treatment. Previous treatment interruptions were more common in subjects with CSF escape. CPE-rank was not a significant predictor of detectable CSF virus or CSF neopterin levels. Conclusions Viral escape in CSF is more common than previously reported, suggesting that low-grade CNS infection may continue in treated patients. Although these findings need extension in longitudinal studies, they suggest the utility of monitoring CSF responses, as new treatment combinations and strategies modify clinical practice.

The role of B, CD4 M T and CD8 M T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated.

Herpes simplex virus (HSV) type 1 was used to infect cultures of human embryonic dorsal root ganglion cells. Infected cultured were studied by electron microscopy. Viral nucleocapsids were observed to be internalized into neuronal cells bodies and neuritic extensions by fusion of the viral envelope and the plasma membrane. No signs of internalization by endocytosis were noted. Nucleocapsids were transported in neurites and were within 2 hrs postinfection found located near the microtubules and close to the nuclear pores in the perikaryon. A primary envelopment of nucleocapsids occurred at the inner lamina of the nuclear membrane and virions appeared between the two laminae. Presence of non-enveloped nucleocapsids outside the nuclear membrane and in close contact with the endoplasmic reticulum suggested that nucleocapsids could pass to the cytoplasmic side probably by de-envelopment at the outer nuclear membrane. A secondary envelopment occurred at the endoplasmic reticulum where the virions also became enclosed in transport vesicles. Enveloped virus appearing in the cytoplasm of neurons and neuritic extensions was always found only inside these transport vesicles. During their passage through the cytoplasm the virion-transport vesicle complexes were surrounded by smaller lysosome-like vesicles possibly derived from the Golgi apparatus. Fusion reactions between vesicles with virions and the smaller vesicles seemed to occur. We discuss if in this way the virion-transport vesicle complexes might be provided with glycosyl transferases and substrates necessary for maturation and completion of glycosylation of the viral envelope glycoproteins. The transport vesicles seemed essential for egress of virions from the infected cell by releasing virus when fusing with the plasma membrane.

It has been suggested that heparan sulphate has a receptor function in the initial phase of the attachment of herpes simplex virus (HSV) to cells. We have studied the influence of glycosaminoglycans on cell adsorption of, and plaque formation by, HSV-1 and HSV-2, with regard to the role of saccharide structure, chain length and charge density. Heparin and highly-sulphated heparan sulphate (1-5 sulphate groups/disaccharide unit), but neither chondroitin sulphate nor dermatan sulphate, were found to compete with the cellular receptor for attachment of HSV. Heparan sulphate preparations of low sulphate content (0.5 and 0.7 sulphate groups/disaccharide unit) failed to show any significant interaction with HSV. Oligosaccharides generated by partial deaminative cleavage of heparin were used to determine the minimum molecular size required for the binding of virus; the smallest oligosaccharide which reacted with HSV was composed of l0 monosaccharide units. The importance of charge density was demonstrated more directly by subfractionation of the heparin dodecasaccharide fraction by anion-exchange HPLC. The virus-binding capacities of the four resulting dodecasaccharide subfractions increased from the least sulphated to the most heavily sulphated fraction. The results reported are discussed in relation to virus-receptor interactions involved in the attachment of HSV, including the reported binding of HSV to the fibroblast growth factor receptor.

SUMMARYAttachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV

SUMMARYHerpes simplex virus type 1 and a fluorescein-labelled lectin (wheat germ agglutinin) were selectively transported to nerve cell bodies located in the inner compartment of a two-chamber tissue culture system after the application of virus or lectin to the neuritic processes in the outer culture compartment. Taxol, which stabilizes and alters intracellular arrangements of microtubules, and nocodazole, which disrupts microtubules, both inhibited this retrograde axonal transport of viral particles and lectin. The transport was also inhibited by erythro-9-3-(2-hydroxynonyl)adenine (EHNA), which blocks ATPases. However, EHNA was also an effective inhibitor of infection with the virus in non-neuronal cells (GMK AH-1). The nature of the action(s) of EHNA on neuritic transport of the virus is therefore less clear.The pathogenesis of herpes simplex virus (HSV) infections involves axonal transport of virus and viral subunits by sensory neurons in the anterograde as well as the retrograde direction. Neurites of dissociated dorsal root ganglion cells have the capacity to internalize the virus (Ziegler & Herman, 1980), presumably by fusion of the viral envelope with the neuritic plasma membrane (Lycke et al., 1984). Since nucleocapsids are detectable in neurites exposed to HSV and are seen around nuclear pores in HSV-infected cell lines (Batterson et al., 1983), we assume that they are involved in retrograde axonal transport of the virus. The mechanisms of this axonal transport of HSV remain to be clarified.We now report the effects on neuritic transport of HSV of three substances capable of inhibiting intracellular transport of endosomes, namely nocodazole, which causes disruption of microtubules, and taxol, which promotes assembly of microtubules (Herman & Albertini, 1984); the third drug, erythro-9-3-(2-hydroxynonyl)adenine (EHNA) (Bouchard et al., 1981) is supposed to inhibit the function of the microtubule-associated ATPase dynein and can inhibit the retrograde movement of organelles in axons. EHNA is considered to have less effect on anterograde axonal transport (Forman et al., 1983).We have used a two-chamber culture system allowing us to infect the neuritic extensions of rat sensory neurons cultured in vitro, without exposing the cell bodies to the virus. For evaluation of the results the neuritic transfer of HSV was compared with the transport of wheat germ agglutinin (WGA).The two-chamber system employed has been described previously (Ziegler & Herman, 1980). Briefly, 35 mm collagen-coated plates were scratched on the bottom, fitted in the centre with an 8 mm cloning cylinder kept in place by silicone high vacuum grease and seeded in the cloning cylinder with trypsin-dissociated dorsal root ganglion (DRG) cells. The cells were obtained by dissecting eight to ten embryos of 16 to 17 day pregnant Sprague-Dawley rats. Trypsinization was performed for 30 min at 37 °C using 0.25~ trypsin (porcine pancreas, type 2, Sigma) in a Ca z÷-and MgZ+-free Hanks' buffer pH 7.2. For the first 2 days, the cells were cultured ...

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-l-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.