Fusing structure and function: a structural view of the herpesvirus entry machinery.

Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.

Reevaluating herpes simplex virus hemifusion.

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a "hemifusion" intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins-glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)-to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins-gD, gB, and gH/gL-were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

Insertion mutations in herpes simplex virus 1 glycoprotein H reduce cell surface expression, slow the rate of cell fusion, or abrogate functions in cell fusion and viral entry.

Of the four required herpes simplex virus (HSV) entry glycoproteins, the precise role of gH-gL in fusion remains the most elusive. The heterodimer gH-gL has been proposed to mediate hemifusion after the interaction of another required glycoprotein, gD, with a receptor. To identify functional domains of HSV-1 gH, we generated 22 randomized linker-insertion mutants. Analyses of 22 gH mutants revealed that gH is relatively tolerant of insertion mutations, as 15 of 22 mutants permitted normal processing and transport of gH-gL to the cell surface. gH mutants that were not expressed well at the cell surface did not function in fusion or viral entry. The screening of gH mutants for function revealed the following: (i) for wild-type gH and some gH mutants, fusion with nectin-1-expressing target cells occurred more rapidly than with herpesvirus entry mediator (HVEM)-expressing target cells; (ii) some gH mutants reduced the rate of cell fusion without abrogating fusion completely, indicating that gH may play a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion; and (iii) only one gH mutant, located within the short cytoplasmic tail, completely abrogated function, indicating that the gH cytoplasmic tail is crucial for cell fusion and viral infectivity.

Innate immunity in HIV infection: enhanced susceptibility to CD95-mediated natural killer cell death and turnover induced by HIV viremia.

In the present study, we performed DNA microarray analyses and phenotypic and functional analyses in an effort to elucidate the mechanisms by which ongoing HIV replication affects the physiologic function of natural killer (NK) cells. Functional assays confirmed an increased propensity of NK cells from HIV-infected viremic individuals to undergo Fas-mediated apoptosis but not CD16- or NKG2D-mediated apoptosis. Serum levels of sFasL and expression of Ki67 on NK cells were markedly elevated in HIV-infected viremic individuals when compared with those of HIV-infected aviremic and HIV-seronegative individuals. Our data demonstrate that ongoing HIV replication results in profound NK-cell abnormalities that are likely to be attributable to the effects of virus-induced immune activation. Of note is an increased susceptibility to cell death mediated by CD95-sFasL interactions. In addition, these NK cells, particularly the CD56(dim) CD16(bright) subset, undergo enhanced cell turnover in vivo, as demonstrated by intracellular Ki67 expression.

Innate immune dysfunction in HIV infection: effect of HIV envelope-NK cell interactions.

We have previously described a number of NK cell dysfunctions in HIV-viremic individuals. In the present study, we performed DNA microarray analysis followed by phenotypic and functional characterization in an effort to investigate which HIV envelope glycoproteins (gp120) affect the physiologic functions of NK cells. Upon treatment of NK cells with HIV gp120, DNA microarray analyses indicated up-regulation of several categories of genes that are associated with apoptosis, suppression of both cellular proliferation and survival, as well as down-regulation of genes that play a vital role in cell proliferation, innate immune defense mechanism, and cell survival. Both subtypes of gp120 suppressed NK cell cytotoxicity, proliferation, and the ability to secrete IFN-gamma. NK cells exposed to X4-subtype HIV gp120 showed a significant decrease in the levels of CC chemokines, while exposure to R5-subtype HIV gp120 had minimal effect. Extended exposure to HIV gp120 resulted in apoptosis of NK cells, further validating the microarray data. Our data demonstrate that exposure of NK cells to HIV envelope proteins results in profound cellular abnormalities at the level of gene expression as well as generic cell functions. These findings are likely to be a consequence of a direct HIV gp120-mediated effect on NK cells. Identification of specific surface receptors on NK cells that interact with HIV envelope proteins might explain how HIV is capable of circumventing innate immune defense mechanisms and establishing infection in susceptible individuals.

Hepatitis B & hepatitis C in HIV-infection.

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the three most common chronic viral infections seen in the world. All three viruses share modes of transmission and hence co-exist in the same host at significantly high rates. HIV-induced immunosuppression has deleterious effects on the natural history, pathophysiology, diagnosis, therapeutic responses to hepatitis viruses. Responses to HBV vaccination are impaired in persons with HIV infection. Co-infection with the hepatitis viruses and HIV is likely to become a major health care catastrophe in the coming years. This review discusses the current trends in the understanding of the biology of co-infection and implications for treating these viruses effectively.