We have two goals in this review: the first is to relate what has been learned about DNA replication from the study primarily of herpes simplex virus type 1 (HSV-1); the second is to note briefly facets of this virus’s mode of DNA replication that might serve as points of intervention for novel chemotherapeutic approaches in order to deal with primary and recurrent herpesvirusinfections in man and animals. The genes involved in S-adenosylmethione and homocysteine metabolism are depicted here. Cytokines, generated by bacterial attachment to Toll receptors or other inflammatory processes are able to reroute tryptophan to serotonin metabolism via effects on indoleamine 2,3-dioxygenase (INDO) resulting in the formation of kynurenic acid. Seroprevalence of Herpes Simplex Virus Type 1 and Type 2 and Coinfection With HIV and Syphilis: The First National Seroprevalence Survey in Saudi Arabia. Finally, we discovered that production of human interleukin-10 (IL-10) and IL-4 partially contributed to HSV-1-induced KSHV replication. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-γ, but not IL-4 cytokine production. Activities of both KSHV LT1/LT2 and huCYC D1 luciferase promoter reporters transfected into NIH 3T3 cells increase 11- and 4-fold, respectively, after release from cell cycle arrest by serum starvation.
The observation that 4a and 4c can be phosphorylated as late as 24 h after infection, i.e., long after their synthesis ceases, suggests that all three forms may have defined functions that persist throughout the reproductive cycle. 1, Murata, Sato, Kimura, Rev Med Virol 2014). In KS lesions, latent KSHV predominates with a low percentage of cells exhibiting lytic replication; the viral genome persists as an episome and has highly restricted expression of latent genes. The latent phase is essential for the development of KSHV-induced malignancies (Staskus et al., 1997). Transmission from the PNS to the CNS occurs across synapse-linked neurons, and the resulting self-amplifying circuit-specific spread has been exploited to trace neural connections in the vertebrate nervous system (47, 158). In parallel, gM is found in various intracellular locations at different moments, ranging from nuclear membranes, perinuclear virions, the TGN, cell surface, and mature extracellular virions. All were positive by LightCycler PCR (DNA polymerase gene target); seventy-five specimens were positive exclusively by the LightCycler assay.
The second transcriptional unit yields an mRNA that encodes a protein designated US3.5 containing residues 77–481 of the US3 PK. The E2F family of transcription factors is cell cycle regulatory proteins that interact with Rb family members (Rb, p107, and p130) (Harbour & Dean, 2000). However, who and how to reactivate latent virus are not well defined. The frequent occurrence of KS in patients with human immunodeficiency virus type 1 (HIV-1) and in transplant recipients strongly suggest that immunodeficiency was a significant factor contributing to the diseases associated with KSHV (Beral and Newton, 1998; Mendez et al., 1999). Movement into the cell from the peripheral site of infection to the nucleus or perinuclear region where most replication takes place is thus accomplished more quickly. Of these sites, phosphorylation of Ser67, and particularly Ser457 by Akt/protein kinase B (Akt/PKB), correlates with nuclear import, destabilization, and decreased inhibition of activator protein-1 (AP-1) mediated transcription (16). Indeed, a couple of agents such as human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) have been proved to be cofactors activating KSHV (Vieira et al., 2001; Lu et al., 2005).
On one hand, co-infection of herpes simplex virus type 1 (HSV-1) and KSHV were frequently detected in AIDS or KS patients (Casper et al., 2002; Chen and Hudnall, 2006; Kumar et al., 2007), recurrent aphthous ulceration patients (Lin et al., 2005), and even in healthy individuals (Miller et al., 2005). On the other hand, HSV-1 could also infect B cells and human vascular endothelial cells, the precursor of KS (Key et al., 1990; Lamontagne and Jolicoeur, 1994). Although HSV-1 and KSHV are not found in similar anatomic compartments during their latent infection, frequent reactivation of latent HSV-1 occurred in AIDS or AIDS-KS patients, leading to appearance of HSV-1 viraemia (Birek and Ficarra, 2006). Viraemia is not only present in the peripheral blood of immunocompromised adults and in neonates, but also during primary herpetic gingivostomatitis in immunocompetent children at the relatively high frequency of 34%, as tested by polymerase chain reaction (PCR) (Harel et al., 2004). Inhibition of virion-induced IE gene expression by Roscovitine.Previous findings have suggested the possibility that cdks are important for expression of viral IE genes (25,26). As M15 protein has been reported to have an exclusively nuclear distribution (22), we cannot rule out a degree of nuclear contamination during extract preparation contributing to the M15 protein cosedimenting with mRNP fractions. HSV-1 is a ubiquitous virus that infects the majority of the human population (approximately 60–90%) (van Benthem et al., 2001).
After primary infection, HSV-1 persists in the host in a latent form and has the potential to cause disease upon reactivation, particularly in the immunocompromised host (Mann et al., 1984; Chen et al., 1998). Epidemiological data showed that in the USA 16–20% of AIDS patients are positive for HSV-1, while 20–57% for KSHV (Krzyzowska et al., 2000; Miller et al., 2006). To address the role of HSV-1 in KSHV replication and KS or AIDS-KS pathogenesis, in this study we performed kinetic studies of KSHV replication induced by HSV-1. HSV-2 is 3 times higher among HIV-infected adults compared to the general population. These novel findings are believed to be the first report on the mechanisms of KSHV activation by HSV-1 and shed light on the understanding of AIDS-KS pathogenesis. A second intratumor DISC/mGM-CSF injection was given 2 days later. In this study, we examined the KSHV LT1 and LT2 transcripts by cDNA cloning, 5′ rapid amplification of cDNA ends (5′ RACE), and primer extension analyses.
On the other hand, most spindle cells, which harbour KSHV in the latent phase replication, undergoing lytic replication are uniformly present in tumours and have been postulated to play a critical role in the maintenance of the tumour. Therefore, the identification of cofactors that enhance KSHV lytic replication at both early and late time points following infection is very important in understanding KS or AIDS-KS pathogenesis. In this study we investigated the kinetic of KSHV replication by HSV-1 and explored the possible mechanisms by which HSV-1 activates KSHV cycle replication. Our experiments provide the direct experimental evidence that HSV-1 can be a potential cofactor of KS. One factor that appears to specifically govern viral transmission into the nervous system without loss of viral replication at sites of peripheral inoculation is the PRV deubiquitinase activity housed within the amino terminus of the pUL36 (VP1/2) tegument protein (17, 89). For instance, Vieira et al. Nevertheless, the cumulative direct costs of the two assays differed by only $1.30.
As shown in Fig. However, silencing of E2F1 did not dramatically increase apoptosis or cell death. During the KSHV replication, the molecular switch that controls the transition from KSHV latency to lytic replication is the product of the ORF50 gene. ORF50 is an immediate-early KSHV gene product whose expression is detectable prior to that of early lytic gene products. Isolated, KI-stripped organelles move on purified actin filaments in vitro [20,21]. (ii) Significantly lower amounts of cleaved PARP product were detected in cells transfected with siRNA and then infected with d120 mutant than in cells mock-treated or transfected with control RNA before infection (compare lanes 1, 2, and 3). Of course, our results did not eliminate the possibility that other immediate-early KSHV genes and their promoters or soluble factors produced by or in response to HSV-1-infected PEL cell lines may also be involved in this process.
In the current study, we found that HSV-1 not only elevated the productions of IL-4 and IL-10, but also promoted mRNAs expression of their receptors in BCBL-1 cells. This enhancement was mediated by HSV-1 production binding to promoters of IL-4, IL-10, and their receptors. IL-10 has been shown to not only serve as a growth factor for AIDS-related B-cell lymphoma (Benjamin et al., 1992; Masood et al., 1995), but also be released and used by PEL cells for autonomous proliferation and was critical to the development and progression of PEL (Drexler et al., 1999; Jones et al., 1999; Oksenhendler et al., 2000). Similarly, IL-4 and its receptor have been found expressed by KS or AIDS-KS tumour cells. Kinetics of Roscovitine-dependent inhibition of IE gene expression. Total RNA was extracted either immediately or after 12 h of incubation. Once HSV-1 infects individuals of KS or AIDS-KS, it can elevate production of Th-2 cytokines, such as IL-10 and IL-4.
On one hand, IL-10 and IL-4 can prevent deletion of HSV-1 and/or KSHV from body by inhibiting Th-1 cytokines-mediated cell immune response. On the other hand, activation of IL-10 and IL-4 can induce KSHV replication, leading to release of many mature KSHV particles, which subsequently infect neighbour B lymphocytes, monocytes and the endothelial cells. Here, by adding rhIL-10 to the culture, we provided direct experimental evidence that IL-10, at least in part, contributed to KSHV lytic replication in BCBL-1 cells, although we did not find that rhIL-4 induced KSHV replication in BCBL-1 cells. Previous studies have shown that Th-1 cytokine IFN-γ from HIV-1-infected T cells or produced by HHV-6-infected BCBL-1 cells was partially responsible for KSHV reactivation (Mercader et al., 2000; Lu et al., 2005). In this study, although IFN-γ was significantly elevated in HSV-1-infected BCBL-1 cells, the results from antibodies blocking assay indicated that it did not contribute to KSHV replication, suggesting a major role in mediating cell immune response against HSV-1 and/or reactivated KSHV, rather than in viral replication. The cells were collected, washed twice, and resuspended in serum-free RPMI at 1 × 107 cells/ml and stored on ice until required. A probe from the late lytic ORF25 gene encoding the major capsid protein (MCP) was used as a comparative control: ORF25 (MCP), sense, 5′-GGCGACATTCATCAACCTCAGG-3′; antisense 5′-ATATCATCCTGTGCGTTCACGAC-3′.
As HSV-1 can alter many cytokines profiles and induce multiple signalling pathways, further studies are needed to better understand whether other cytokines and their signals by HSV-1 are also involved in KSHV replication in KS. Fig. S1. HSV-1 activates KSHV lytic cycle replication via ORF50. A recent analysis of tegument protein associations with capsids based on resistance to detergent and salt extraction classified seven proteins as components of the inner tegument: pUL14, pUL16, pUL21, pUL36 (VP1/2), pUL37, pUs3, and ICP0 (139). RT-PCR analysis for ORF50 mRNA in si-constructs-transfected BCBL-1 cells infected with HSV-1. ORF50 mRNA expression in si-1 (si-1), si-2 (si-2), si-3 (si-3), si-vector (si-v)-transfected or PBS-treated BCBL-1 cells further infected with HSV-1 for 12 h was detected by RT-PCR.
The arrow points to the cleavage product of PARP characteristic of apoptosis. RS cells were infected with BHV-1 or HSV-1 using a moi of 5 and whole cell lysate collected at the designated times post-infection. ORF26 mRNA expressed in si-2-transfected BCBL-1 cells following infection with HSV-1. ORF26 mRNA expression in si-vector-transfected BCBL-1 cells infected with Mock (Mock + si-vector), si-vector-transfected BCBL-1 cells infected with HSV-1 (HSV-1 + si-vector), and si-2-transfected BCBL-1 cells infected with HSV-1 (HSV-1 + si-2) for 3, 6, 12, 24, 48 and 72 h was quantified by real-time quantitative PCR. Microtubules are also formed from basal bodies at the cortex which produces flagella and cilia. At 24 h after transfection, the cells were mock-treated or exposed to 10 μM LMB. Fig.
S2. HSV-1 infection results in changes of cytokine protein expression profile in BCBL-1 cells. A. A representative experiment is shown as dot blots representing the protein expression profile of selected cytokines. (3). It is likely, however, that this region mediates an interaction between ICP27 and a component of the translation machinery. The results are given as ratio of protein expression of HSV-1 infected to that of Mock infected controls (see Table 2).
B. Selected cytokines are listed. The selected cytokines are listed or indicated at the supplier’s website (http://www.raybiotech.com/map/human_5_map.pdf). Pos and Neg represent positive and negative controls respectively. Fig. The DISC/mGM-CSF virus (dH2B) is thymidine kinase negative, HSV envelop glycoprotein negative, and expresses mGM-CSF following infection of normal or complementing cells. Northern blot analysis.Probes internal to ORF72 (vCYC) (Fig.1A) and ORF K13 (vFLIP) (Fig.
Real-time quantitative PCR was used to detect relative quantities of ORF26 mRNA in Mock-infected BCBL-1 cells plus 13.32 μg ml−1 of goat control IgG (Mock + Cont IgG), HSV-1-infected BCBL-1 cells plus 13.32 μg ml−1 of goat control IgG (HSV-1 + Cont IgG), HSV-1-infected BCBL-1 cells plus 13.32 μg ml−1 of pAb against IL-4 (HSV-1 + pAb-IL-4) for 3, 6, 12, 24 and 48 h indicated. The results from three independent experiments performed in triplicate are shown. Fig. S4. Transport occurs along microtubules at rates in excess of 1 μm s−1 (9, 80, 168). Real-time quantitative PCR was used to detect relative quantities of ORF26 mRNA in Mock-infected BCBL-1 cells plus 3.33 μg ml−1 of goat control IgG (Mock + Cont IgG), HSV-1-infected BCBL-1 cells plus 3.33 μg ml−1 of goat control IgG (HSV-1 + Cont IgG), HSV-1-infected BCBL-1 cells plus 3.33 μg ml−1 of pAb against eotaxin-3 (HSV-1 + pAb-IL-eota) for 3, 6, 12, 24 and 48 h indicated. The results from three independent experiments performed in triplicate are shown.
US3 shuttles between the nucleus and cytoplasm. As a control for nuclear proteins, we examined histone H3 and found, as expected, that histone H3 was detected only in the nucleus after infection of RS cells with HSV-1 or BHV-1 ().