Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Human submandibular saliva inhibits human immunodeficiency virus type 1 infection by displacing envelope glycoprotein gp120 from the virus.

Human submandibular saliva reduces human immunodeficiency virus type 1 (HIV-1) infection in vitro. To define the mechanism of inhibition, virus was incubated with saliva or medium, velocity sucrose gradient centrifugation was performed, and fractions were analyzed for p24 and gp120. The results show that after incubation with saliva, the envelope glycoprotein was displaced from both a laboratory-adapted and a low-passage clinical HIV-1 isolate. To identify the salivary protein(s) responsible, submandibular saliva was fractionated by anion- exchange chromatography. Protein fractions containing anti-HIV activity were assayed for their ability to strip gp120 from virus. The partially purified active fractions contained two high-molecular-weight sialyated glycoproteins identified as salivary agglutinin and mucin, as well as several lower-molecular-weight proteins. It thus appears that specific salivary proteins interact with HIV-1 to strip gp120 from the virus with a resultant decrease in infectivity.

Construction of attenuated HIV-1 accessory gene immunization cassettes.

Delivery of genetic expression cassettes into animals can effectively induce both humoral and cellular immunity to the expressed gene product. Previously, we used this strategy to immunize against HIV-1 structural and enzymatic proteins in mice, non-human primates and in humans. In contrast, the use of the accessory genes including vif, vpr, vpu and nef as immunotherapeutic vaccine targets has not been well characterized. Our goal is to design an effective genetic HIV vaccine, which includes the accessory genes as part of a multi-component immunogen. In order to develop accessory genes as genetic vaccines, we have molecularly cloned and analysed the sequence variation and immunogenic potential present in these genes derived from viral isolates obtained from HIV-1 infected patients and laboratory isolates. Prototype genetic variants were selected and their ability to induce humoral and cellular immune responses was studied in animal models. We observed that attenuated accessory genes can effectively induce both humoral and cellular responses in mice and the resulting immune response is directly correlated with DNA concentrations delivered and the number of boosts. This strategy can be used generally to develop an effective, safe DNA vaccine for any pathogen.

Submandibular salivary proteases: lack of a role in anti-HIV activity.

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.

Viral interference with antibody and complement.

Viruses have evolved strategies to evade immunity mediated by antibody and complement. Herpesviruses and coronaviruses encode IgG Fc binding proteins that inhibit IgG activity, enabling the virus or infected cell to escape antibody attack. Herpesviruses, vaccinia virus and HIV-1 have the capacity to interfere with complement, either by incorporation of cellular complement regulatory proteins into the virion envelope or cell membrane, or by expression of viral molecules that mimic functions of complement regulatory proteins. The structure and biological activities of herpes simplex virus type 1 (HSV-1) glycoproteins gE, gI and gC are described. These glycoproteins protect HSV from immune attack; HSV-1 gE/gI form a complex that binds the Fc domain of IgG while gC is a C3b binding complement regulatory protein, providing a survival advantage to the virus in vitro and in vivo by inhibiting immune functions.

In vivo immune evasion mediated by the herpes simplex virus type 1 immunoglobulin G Fc receptor.

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcgammaR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcgammaR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcgammaRs. NS-gE339 expresses gE and gI, is FcgammaR-, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcgammaR does not bind murine IgG; therefore, the absence of an FcgammaR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcgammaR- mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcgammaR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcgammaR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.

Development of genetic vaccines for pathogenic genes: construction of attenuated vif DNA immunization cassettes.

OBJECTIVE: To develop a putative immunization cassette using HIV-1 vif accessory gene derived from HIV-1 clinical specimens as a component of a DNA vaccine for HIV-1. METHODS: vif genes were cloned from HIV-1-infected patients and the sequence variation present within the patients was analyzed. Prototypic genetic variants were selected and the ability of these clones to induce humoral and cellular immune responses was studied in animals. The selected protective genetic variants were biologically characterized through transcomplementation assays using primary cells infected with a vif-defective HIV-1 proviral clone. RESULTS: Analysis of vif variants from different patients revealed that vif is highly conserved with the open reading frame remaining intact in vivo. It was shown that attenuated vif clones from HIV-1-infected subjects can effectively induce both humoral and cellular responses against Vif protein in mice. Evaluation of the cellular responses in vitro using human cellular targets infected with a clinical HIV-1 isolate showed that vif clones could induce cellular responses capable of destroying the virus. CONCLUSIONS: The vif variants developed in this study exhibited non-productive phenotypes, yet were capable of inducing specific immune responses against HIV-1. These constructs could be used as part of a DNA vaccine strategy for HIV-1. This vaccine adaptation strategy could be used for the development of immunogens for any pathogen resulting in cross-reactive immunity and attenuated gene pathogenesis.

HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro.

Among the putative accessory genes of HIV-1, the 96-amino-acid virion-associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic-to-nuclear translocation, which empowers HIV to infect and replicate in non-dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV-1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic-to-nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti-glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti-HIV drug development and has significance for HIV-1 disease progression.

The herpes simplex virus-1 glycoprotein E (gE) mediates IgG binding and cell-to-cell spread through distinct gE domains.

Herpes simplex virus-1 (HSV-1) glycoprotein E (gE) is a multifunctional protein capable of both binding the Fc portion of IgG and mediating cell-to-cell spread of HSV-1. Here we report that the domain on gE involved in IgG binding is distinct from the domain involved in mediating cell-to-cell spread. To do this we have used five mutants of the HSV-1 strain NS: NS-gE(null), a gE deletion virus; rNS-gE(null), a gE rescued virus; NS-gE339, a gE mutant virus with a four amino acid insert at position 339; rNS-gE339, a gE rescue of NS-gE339; and NS-gE406, a gE mutant virus with the same four amino acids inserted at position 406. Using IgG coated sheep red blood cells in rosetting assays, we show that the NS-gE339 does not bind IgG, yet retains the ability to mediate normal cell-to-cell spread. These results demonstrate that the gE domain involved in IgG binding differs from the domain involved in cell-to-cell spread.

Inhibition of HIV infectivity by human saliva.

OBJECTIVE: Human saliva is known to decrease HIV infectivity in vitro. The purpose of this study was to extend these findings and to focus on the mechanism of action of these salivary factor(s). DESIGN: A number of viruses and several assay systems have been utilized to determine if the effect of submandibular saliva is directly on the virus, on the host cell, or on the virus-cell interaction. MATERIALS AND METHODS: Submandibular saliva from seronegative donors was incubated with HIV-1, other retroviruses, or unrelated viruses. Viral infectivity was monitored either by determining p24 antigen levels in peripheral blood mononuclear cells or Sup T1 cells, or using HeLa cells expressing CD4 and an HIV derived long terminal repeat linked to the beta-galactosidase gene. RESULTS: The inhibition of viral infectivity by submandibular saliva is specific for HIV-1. While inhibition increases with time of incubation of saliva with virus, pretreatment of cells with saliva does not inhibit HIV production, and saliva has only modest inhibitory effects when added to HIV-infected cells. CONCLUSIONS: It appears that the effect of submandibular saliva on decreasing the infectivity of HIV-1 is directly on the virus, rather than on the host cell.

Human submandibular saliva specifically inhibits HIV type 1.

Studies from a number of laboratories have shown the presence of factor(s) in whole, parotid, and submandibular human saliva capable of inhibiting HIV-1 infectivity in vitro. Data from our laboratory suggested that the level of anti-HIV-1 activity is higher in submandibular than parotid or whole saliva. Previous results obtained with pooled submandibular saliva from seronegative individuals included a filtration step following saliva-virus interaction. In this article, we present data on the HIV-1 inhibitory activity of individual submandibular saliva samples collected from 15 donors. We show that although anti-HIV activity is quantitatively similar in most individuals (9 of 15), some (4 of 15) are much less active than others and some (2 of 15) lack inhibitory activity. We also show that for most individuals the level of anti-HIV inhibitor is similar with or without a filtration step. However, 2 of the 15 samples demonstrated activity only after filtration. The quantitative and qualitative anti-HIV activity of individual saliva samples appeared to reflect differences in the individual donors. We further show that the anti-HIV activity of submandibular saliva is demonstrated not only against laboratory strains of HIV-1 but is similarly active against three clinical HIV-1 isolates. In contrast, submandibular saliva had little effect on the infectivity of HIV-2 or SIV.

Mapping regions of herpes simplex virus type 1 glycoprotein I required for formation of the viral Fc receptor for monomeric IgG.

Glycoprotein E (gE) and glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) form a complex that binds the Fc domain of monomeric IgG. In this study, we used two approaches to map the regions of gI-1 required for formation of the HSV-1 Fc receptor for monomeric IgG. First, we constructed six plasmids encoding gD-1/gI-1 fusion proteins. Each fusion protein contains a large gI-1 peptide inserted into the ectodomain of gD-1. gD-1/gI-1 fusion proteins were coexpressed with gE-1 using a transfection-infection assay in which cells were transfected with individual fusion protein constructs and then infected with a gE+/gI- virus. Cells were then assayed for monomeric IgG binding using immunofluorescence microscopy. Transfection-infection with two of six fusion proteins conferred monomeric IgG binding activity to cells, whereas cells infected with gE+/gI- virus alone failed to bind IgG monomers. The smallest gI-1 peptide to confer monomeric IgG binding activity contained amino acids 43 to 192. To more precisely map the region of gI-1 required for monomeric IgG binding, we constructed a panel of 10 gI-1 linker insertion mutants. Transfection-infection studies identified two mutants containing linker insertions at gI-1 amino acids 128 and 145, which failed to bind monomeric IgG. The other eight mutants demonstrated wild-type IgG binding activity. Taken together, these results indicate that the region of gI-1 between amino acids 128 and 145 is required for formation of the HSV-1 Fc receptor for monomeric IgG.

HIV-1 viral protein R (Vpr) as a regulator of the target cell.

Among the putative 'accessory genes' of HIV-1, the 96 amino acid virion-associated Vpr gene product has been described to have several novel biological activities. These include cytoplasmic-to-nuclear translocation thus empowering HIV to infect and replicate in nondividing cells and to function to increase viral replication, particularly in monocytes. Along with these viral effects, we describe the dramatic biological changes induced by HIV-1 Vpr in the target cells of HIV infection including induction of changes in transcriptional patterns and complete inhibition of proliferation which collectively is termed differentiation. These changes occur in the absence of other viral gene products and suggest that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T-cell lines has been proposed by several groups to demonstrate that the inhibition of proliferation specifically arrests the cell cycle further supporting the notion that Vpr activity is directed at cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, a pathway involved in the regulation of the state of the cell in cytoplasmic-to-nuclear translocation and in the modulation of host cell transcription. Importantly, certain antiglucocorticoids have been shown to modulate Vpr activity in vitro. These results demonstrate that the cell contains specific receptor(s) molecule(s) through which Vpr mediates its activity and that these molecules have implications for cell biology in general. These results collectively demonstrate that Vpr represents a unique target for anti-HIV drug development and has significance for HIV-1 disease progression.

Differential production of interleukin 10 during human immunodeficiency virus infection.

Interleukin 10, a product of T and B cells and monocytes, displays many Th2-like properties through inhibition of Th1 cell functions. Interleukin 10 is thought to play a major role in the immune dysfunction seen in HIV-infected individuals. In this study, we evaluated in detail the production of IL-10 during HIV infection. Although the constitutive production of IL-10 did not differ in PBMCs from healthy donors and HIV-infected individuals, IL-10 was differentially produced in response to polyclonal activators. The overall plasma IL-10 levels were similar in 32 controls and 67 patients at different stages of the disease and receiving different antiretroviral drugs. However, patients with low CD4 T cell count (< 200/mm3) secreted approximately three-fold more IL-10 than did patients with high CD4 T cell count (> 500/mm3). Competitive/quantitative PCR revealed similar levels of mRNA expression in PBMCs from controls and HIV-infected individuals. In vitro HIV infection rapidly and transiently induced IL-10 production in PBMCs and monocytes, and the low level of endogenously secreted IL-10 failed to inhibit HIV replication in acutely infected monocytes. On the other hand, HIV infection of selected CD4+ T cell clones generated in a Th1- or Th2-like environment, differentially up-regulated IL-10 production, with significantly higher production by Th2 clones. Together, our data indicate that IL-10 production is more complex than previously thought, and may depend on several factors such as producer cells, nature of the stimuli, as well as viral isolates.

Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization.

The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.

Structure-function studies of HIV-1: influence of long terminal repeat U3 region sequences on virus production.

DNA sequence analyses of several human immunodeficiency virus (HIV) isolates revealed extensive genetic diversity in the env gene and, to a lesser extent, in other regions of the viral genome, including the long terminal repeat (LTR) sequences. Since the LTRs contain elements responsible for the control of transcription, the difference in the LTR region may play a crucial role in the overall replication rate of HIV. To evaluate the role of the LTR, we have constructed a number of infectious hybrid HIV molecular clones containing LTRs from different proviral DNAs linked to the body of the viral genome, and analyzed them in a transient expression system. Both parental and hybrid proviral DNAs were transfected into human rhabdomyosarcoma cells for monitoring virus production. Proviral DNA designate pZ6 (HIVZr6) showed a high level of virus in the medium of the transfected culture in comparison to the pHXB2 (HIVHTLV-III) and pARV (HIVSF-2) DNAs. Hybrid proviral DNAs containing viral genes from pZ6, linked to LTR U3 sequences of pHXB2 and pARV at the 5' end, showed virus production similar to the levels observed with pZ6. These results indicate that the extent of virus production does not correlate with the LTR U3 sequences, and may involve other regions of the viral genome.

Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity.

In an effort to understand the contribution of the primer-binding site (PBS) region to human immunodeficiency virus (HIV) replication, we have constructed a mutant HIV proviral DNA with an alteration in the 5' end of the PBS. The PBS mutant proviral DNA was characterized by transfection of the viral DNA into CD4+ and non-CD4+ target cells. The results indicate that mutation in the PBS reduced the level of viral particles released into the medium of transfected cells in comparison to wild-type proviral DNA. The viral particles were noninfectious upon transmission to established CD4+ cell lines and phytohemagglutinin-stimulated peripheral blood lymphocytes. Electron microscopic analysis of the transfected cells revealed no abnormalities in the structure of the virion directed by the mutant proviral DNA. Also, the protein and RNA contents of the mutant virions were similar to the wild type. The quantitation of intracellular viral structural protein in the transfected cells, however, indicated that the PBS mutation may have an effect on the assembly of viral particles in addition to completely abolishing reverse transcription of viral RNA into DNA. These results provide evidence that the PBS region of the viral genome has multiple functions in HIV-1 replication.

Analysis of the viral determinants underlying replication kinetics and cellular tropism of human immunodeficiency virus.

Human immunodeficiency viruses (HIVs) isolated from infected individuals show genetic and biological diversity. To delineate the genetic determinants underlying specific biological characteristics such as rate of replication and cellular tropism, generation of hybrid HIV using viruses which exhibit distinct biological feature is essential. We have used three different infectious HIV proviral DNAs, designated pZ6, pHXB2 and pARV, derived from HIVZr6, HIVHTLV-IIIB and HIVSF-2 strains, respectively, to construct hybrid HIV. Proviral DNAs differed in their ability to direct the synthesis of viral particles upon transfection into cells and the viruses derived from the molecular clones exhibited different cellular tropism. Three different methods were utilized to generate hybrid HIV, including construction of hybrid proviral DNA using molecular techniques, intracellular ligation of viral DNA fragments and the homologous recombination approach. The chimeric proviral DNAs with exchanges involving only the long terminal repeat (LTR) region indicated that LTR does not exert influence on the overall level of virus production despite extensive differences in the U3 region of the LTR. Regarding the cellular tropism of HIV, the virus derived from pHXB2 productively infected CEMx174 cells. On the other hand, pARV-derived virus did not show productive infection of CEMx174 cells. The hybrid HIV containing the 3'-end of the genome from pARV and the 5'-end of the genome from pHXB2 was effective in infecting CEMx174 cells. However, the converse hybrid containing the 5'-pARV and the 3'-pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef may play a role in the ability of virus to infect a certain cell type.

Structure and expression of the beta-platelet-derived growth factor receptor gene in human tumor cell lines.

Southern digests of DNA from 15 human connective tissue tumor-derived cell lines (and cell strains) and from peripheral blood lymphocytes from normal volunteers were prepared to compare structure of the beta-platelet-derived growth factor receptor gene in normal and tumor-derived cells. The tumor-derived samples had no alterations in gene structure, nor was gene amplification evident. Two restriction fragment length polymorphisms were detected within the beta-receptor gene. Expression of the alpha- and beta-platelet-derived growth factor receptor was quantified using Northern blots. Expression was not tumor-type specific. For example, one rhabdomyosarcoma cell line expressed only the alpha-receptor, whereas two others expressed the beta. In contrast, a human fibroblast cell strain expressed both receptor types. Alterations in receptor expression may be a result of the tumorigenic process.