The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro.

Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

Non-human primate model of Kaposi's sarcoma-associated herpesvirus infection.

Since Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi's sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.

The human cytomegalovirus UL112-113 locus can activate the full Kaposi's sarcoma-associated herpesvirus lytic replication cycle.

Human cytomegalovirus (HCMV) infection of a cell containing latent Kaposi's sarcoma-associated herpesvirus (KSHV) results in the activation of KSHV lytic replication and the production of infectious virus. In this study, we examined the HCMV genes identified as having a role in the activation of HCMV early genes for their ability to activate KSHV lytic replication. It was found that the UL112-113 locus was able to activate the complete KSHV lytic cycle, while the UL122-123 locus, encoding the IE1 and IE2 proteins, known to be strong transactivators, did not.

Gamma delta+ T cells involvement in viral immune control of chronic human herpesvirus 8 infection.

Little is known about what effector populations are associated with the control of human herpesvirus 8 (HHV-8) infection in vivo. We compared T lymphocyte subsets among HIV-HHV-8+ and HIV-HHV-8- infected human individuals. alphabeta+ T cells from HHV-8-infected individuals displayed a significantly higher percentage of differentiated effector cells among both CD4+ and CD8+ T cell subsets. HHV-8 infection was associated with significant expansion of gammadelta+ Vdelta1 T cells expressing a differentiated effector cell phenotype in peripheral blood. In vitro stimulation of PBMC from HHV-8-infected individuals with either infectious viral particles or different HHV-8 viral proteins resulted in gammadelta Vdelta1 T cell activation. In addition, gammadelta Vdelta1 T cells displayed a strong reactivity against HHV-8-infected cell lines and prevented the release of infectious viral particles following the induction of lyric replication. These data indicate that gammadelta T cells play a role in both innate and adaptive T cell responses against HHV-8 in immunocompetent individuals.

Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo.

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV, purified HPCs were exposed to KSHV, and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells, and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection, persistence, and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo.

Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation.

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression.

A hallmark of all herpesvirus is the ability to exist in either a latent, or lytic, state of replication, enabling the lifelong infection of its host. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) can efficiently establish a latent infection in a variety of cell types in vitro, making it a valuable model for the study of latency and reactivation. To facilitate the identification of KSHV lytic replication, and allow subsequent experiments with live cells, a recombinant virus, rKSHV.219, was constructed using JSC-1 cells that expresses the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-1alpha promoter, and with the gene for puromycin resistance as a selectable marker. rKSHV.219 from JSC-1 cells was used to infect Vero cells for purification of the recombinant virus. Vero cells were also used for the production of rKSHV.219 at levels of 10(5)-10(6) infectious units (IU) of virus per milliliter using a combination of KSHV/RTA expressed from a baculovirus vector, BacK50, and butyrate. Virus produced from Vero cells was used to infect human fibroblasts (HF), 293, DU145, T24, HaCaT, and HEp-2 cells, and in all cells except 293 cells, only a latent infection was established with GFP expression, but no RFP expression. In 293 cells, 10-15% of cells showed lytic gene expression. Both primary and immortalized microvascular endothelial cells (MVEC) were also infected with rKSHV.219, and reduced spontaneous lytic replication was found in immortalized cells. In all cells used in this study, rKSHV.219 efficiently established latent infections from which the virus could be reactivated to productive lytic replication. This work also demonstrated strong synergy between KSHV/RTA and butyrate for the activation of KSHV lytic replication and the production of infectious virus.

Reduced levels of neutralizing antibodies to Kaposi sarcoma-associated herpesvirus in persons with a history of Kaposi sarcoma.

Kaposi sarcoma-associated herpesvirus (KSHV) has been identified as the etiologic agent of Kaposi sarcoma (KS). Although KSHV is required for the development of KS, immune dysfunction is a common and important cofactor in the development of KS, as illustrated by the presence of KS in association with HIV infection or immunosuppressive-drug treatment after transplantation. Because neutralizing antibodies (NAb) constitute an important component of an antiviral immune response, we examined the functionality of the humoral immune response associated with KS, by measuring KSHV NAb titers in 3 groups of subjects. Group 1 included subjects who were KSHV positive, KS positive, and human immunodeficiency virus (HIV) positive; group 2 included subjects who were KSHV positive, KS negative, and HIV positive; and group 3 included subjects who were KSHV positive, KS negative, and HIV negative. NAb titers were significantly lower among subjects with KS, compared with subjects who were infected with KSHV but who did not have clinical evidence of KS, in a multivariate model adjusted for HIV infection and CD4 T cell count. The data from the present study suggest that NAb may play a role in the control of KSHV infection and the prevention of progression of KSHV infection to KS.

Immunodominance among herpes simplex virus-specific CD8 T cells expressing a tissue-specific homing receptor.

The study of immunodominance within microbe-specific CD8 T cell responses has been challenging. We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides. Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen. The specificity of the resultant cytotoxic clones was determined. Clonal frequencies were compared with each other and with the total number of cytotoxic clones. For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides. Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects. Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots. We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.