Immune evasion strategies of Kaposi's sarcoma-associated herpesvirus.

To establish lifelong infection in the presence of an active host immune system, herpesviruses have acquired an impressive array of immune modulatory mechanisms that contribute to their success as long-term parasites. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently discovered human tumor virus and is associated with the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV has acquired a battery of genes to assist in viral survival against the host immune response. These viral gene products target a variety of host immune surveillance mechanisms, including the cytokine-mediated immune response, apoptosis, natural killer (NK) cell killing and T cell-mediated responses. This review summarizes our understanding of the role of these viral proteins in the escape from host immune surveillance, which ultimately contributes to lifelong infection and pathogenesis of KSHV.

Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine.

Lysophosphatidylcholine (LPC), a component of oxidized low-density lipoprotein, exerts various biological effects on vascular endothelial cells. However, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipitation and Western blotting assays. Treatment of cells with LPC promoted a rapid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phosphorylation was inhibited by calcium chelators, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phosphorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine phosphorylation, and Grb2 binding to Shc and induced Ras activation. These results provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LPC-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Shc/Grb2 signaling.

Inhibition of p21/Waf1/Cip1/Sdi1 expression by hepatitis C virus core protein.

The possibility of interaction between hepatitis C virus (HCV) core protein and the cell cycle regulator protein p21/Waf1/Cip1/Sdi1 (p21/Waf1) in cultured cells was analyzed. Although colocalization of HCV core protein and p21/Waf1 was not clearly observed, p21/Waf1 expression was much weaker in HCV core protein-expressing cells than in the control. A Northern blot analysis showed nearly the same level of p21/Waf1 mRNA in both cells, suggesting that HCV core protein inhibited p21/Waf1 expression post-transcriptionally. The degradation patterns of p21/Waf1 did not differ significantly in HCV core protein-expressing cells and in the control, suggesting that the stability of p21/Waf1, once it was accumulated in the cell, was not significantly affected by HCV core protein. But this does not necessarily exclude the possibility that synthesis, maturation, and nuclear transport of p21/Waf1 is impaired, or that the degradation of newly synthesized, improperly processed p21/Waf1 is promoted by HCV core protein. The decrease in p21/Waf1 accumulation was partially inhibited by proteasome inhibitors and a calpain inhibitor in both HCV core protein-expressing cells and the control. In vitro kinase assay revealed that a p21/Waf1-mediated inhibition of cyclin-dependent kinase 2 activity was partially negated by HCV core protein. Taken together, the present results suggest that HCV core protein inhibits p21/Waf1 expression post-transcriptionally and impairs the function of p21/Waf1 in the cell.

Nuclear localization and intramolecular cleavage of N-terminally deleted NS5A protein of hepatitis C virus.

The full-size NS5A (NS5A-F) of hepatitis C virus is localized in the cytoplasm despite the presence of a functional nuclear localization signal (NLS) in its C-terminal region (amino acids (aa) 354-362). In the present study, we demonstrated that a short stretch of sequence near the N-terminus of NS5A (aa 27-38) masked the functional NLS, preventing NS5A from being transported to the nucleus. This sequence, referred to as an NLS-masking sequence, was distinct from a nuclear export signal, as it did not actively target a protein to the cytoplasm. We also found that other sequences located at either an N- (aa 1-21) or a C-terminal region (aa 353-447) were responsible for targeting NS5A to the cytoplasm. Western blot analysis of the transfected cells revealed that NS5A mutants that had been N-terminally deleted by 66 aa or more were cleaved at a certain cleavage site, generating a common fragment of ca. 40 kDa. This result implies the possible presence of a cleavage site in the NS5A sequence around aa 150, which is exposed through conformational alteration upon the N-terminal deletions.

Inhibition of natural killer cell-mediated cytotoxicity by Kaposi's sarcoma-associated herpesvirus K5 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) K3 and K5 proteins dramatically downregulate MHC class I molecules. However, although MHC class I downregulation may protect KSHV-infected cells from cytotoxic T lymphocyte recognition, these cells become potential targets for natural killer (NK) cell-mediated lysis. We now show that K5 also downregulates ICAM-1 and B7-2, which are ligands for NK cell-mediated cytotoxicity receptors. As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. Conversely, de novo expression of B7-2 and ICAM-1 resensitizes the K5-expressing cells to NK cell-mediated cytotoxicity. This is a novel viral immune evasion strategy where KSHV K5 achieves immune avoidance by downregulation of cellular ligands for NK cell-mediated cytotoxicity receptors.

The collagen repeat sequence is a determinant of the degree of herpesvirus saimiri STP transforming activity.

Herpesvirus saimiri (HVS) is divided into three subgroups, A, B, and C, based on sequence divergence at the left end of genomic DNA in which the saimiri transforming protein (STP) resides. Subgroup A and C strains transform primary common marmoset lymphocytes to interleukin-2-independent growth, whereas subgroup B strains do not. To investigate the nononcogenic phenotype of the subgroup B viruses, STP genes from seven subgroup B virus isolates were cloned and sequenced. Consistent with the lack of oncogenic activity of HVS subgroup B viruses, STP-B was deficient for transforming activity in rodent fibroblast cells. Sequence comparison reveals that STP-B lacks the signal-transducing modules found in STP proteins of the other subgroups, collagen repeats and an authentic SH2 binding motif. Substitution mutations demonstrated that the lack of collagen repeats but not an SH2 binding motif contributed to the nontransforming phenotype of STP-B. Introduction of the collagen repeat sequence induced oligomerization of STP-B, resulting in activation of NF-kappaB activity and deregulation of cell growth control. These results demonstrate that the collagen repeat sequence is a determinant of the degree of HVS STP transforming activity.

Inhibition of intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1.

The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.

Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy.

The signal transduction mechanisms mediating hypertrophic responses in myocardial cells (MCs) remain uncertain. We investigated the role of the extracellular signal-regulated kinase (ERK) cascade in myocardial cell hypertrophy by the strategy of using the adenovirus-mediated overexpression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which is the upstream activator of ERK. We generated recombinant adenoviruses expressing constitutively active MEK1 (MEK1 EE) and dominant negative MEK1 (MEK1 DN). Overexpression of MEK1 EE in MCs activated ERK1/2 and subsequently induced atrial natriuretic peptide (ANP) mRNA expression. In addition, MEK1 EE overexpression resulted in an increase in cell size and sarcomeric reorganization. In contrast, overexpression of MEK1 DN in MCs inhibited endothelin-1 (ET-1)-, phenylephrine (PE)-, leukemia inhibitory factor (LIF)-, isoproterenol (ISP)-, and mechanical stretch-induced ERK activation and ANP mRNA expression. MEK1 DN overexpression inhibited ET-1-, PE-, LIF-, and ISP-induced increases in cell size and sarcomeric reorganization. Consistent with the observed effects on cellular morphology, overexpression of MEK1 EE resulted in an increase in amino acid incorporation, while overexpression of MEK1 DN inhibited ET-1-, PE-, LIF-, ISP-, and mechanical stretch-induced increases in amino acid incorporation. These results indicate that the ERK cascade plays an important role in the signaling pathway leading to the development of myocardial cell hypertrophy.

Viral load in Indonesian patients with chronic liver disease and in blood donors infected with different subtypes of hepatitis C virus.

The viral load of different hepatitis C virus (HCV) subtypes, including the globally distributed HCV-1b and the unique Indonesian subtype HCV-1c, was analyzed using serum samples obtained from Indonesian blood donors and patients with chronic liver disease. The mean viral load of HCV-1c was comparable with that of HCV-1b, suggesting that HCV-1c is as pathogenic as HCV-1b. On the other hand, the mean viral load of HCV-2a was lower than that of HCV-1b or HCV-1c, with this result being consistent with previous observations. Interestingly, some HCV-2a strains were associated with a high viral load that was almost equivalent to that of HCV-1b and HCV-1c. This result implies the possibility that there exists a minor fraction of HCV-2a strains that cause higher levels of viremia compared with the majority of ordinary HCV-2a strains.

Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1.

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.

Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins.

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.

Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway.

Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal-regulated kinase (ERK) in growth factor-stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2-induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-gamma. Taken together, our data demonstrate that ERK activity is required for FGF-2-induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.

Possible Involvement of the NS3 Protein of Hepatitis C Virus in Hepatocarcinogenesis : Its Interaction with the p53 Tumor Suppressor.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped virus, whose genome is single-stranded, positive-sense RNA of approximately 9.5 kb. The viral genome exhibits a considerable degree of sequence variation, based on which HCV is currently classified into at least 6 clades (previously called genotypes) and more than 60 subtypes (1,2). The HCV genome encodes a polyprotein consisting of about 3010-3033 amino acid residues. A number of studies have shown that this polyprotein is cleaved cotranslationally and posttranslationally into mature viral proteins, which are arranged in the order NH(2)-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (3,4).

Complex formation of the nonstructural protein 3 of hepatitis C virus with the p53 tumor suppressor.

By co-immunoprecipitation analysis, we demonstrated that wt-p53 formed a complex with non-structural protein (NS) 3 of hepatitis C virus, both in the absence and the presence of NS4A, a viral cofactor that strongly associates with NS3. Deletional analysis revealed that a portion near the N-terminus of NS3 (amino acids (aa) 1055 and 1200), which is different from the NS4A binding site, was necessary for the complex formation with wt-p53. On the other hand, a portion near the C-terminus of wt-p53 (aa 301-360), which has been reported to contain the oligomerization domain, was important for the complex formation with NS3.

Follow-up study of hypervariable region sequences of the hepatitis C virus (HCV) genome in an infant with delayed anti-HCV antibody responses.

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti-HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti-HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.

Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus.

At present, the mechanism of replication of the HCV genome remains unclear. Recently, NS5B and NS3 of HCV have been shown to exhibit RNA-dependent RNA polymerase and helicase activities, respectively, both of which are indispensable for virus RNA replication. In this study, we examined the complex formation of NS5B with NS3 and NS4A, a cofactor for NS3. We show here that NS5B forms a complex with NS3 through an amino-terminal portion of NS3. The NS3-NS5B complex formation took place both in the presence and absence of NS4A. We also demonstrate that NS5B form a complex with NS4A in the absence of NS3. These results suggest that NS3, NS4A and NS5B interact with each other to form a complex that functions as part of the replication machinery of HCV.

Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization.

Subcellular localization of the NS2 and NS3 proteins of hepatitis C virus was analyzed. In stable Ltk transfectants inducibly expressing an NS2-NS3 polyprotein (amino acids [aa] 810 to 1463), processed full-size NS2 (aa 810 to 1026) was detected exclusively in a cytoplasmic membrane fraction. On the other hand, the other processed product, carboxy-truncated NS3 (NS3 deltaC1463; aa 1027 to 1463), was present in both cytoplasmic and nuclear fractions. To further analyze subcellular localization of NS3, NS3 deltaC1459 (aa 1027 to 1459), full-size NS3 (NS3F; aa 1027 to 1657), and both amino- and carboxy-truncated NS3 (NS3 deltaNdeltaC; aa 1201 to 1459) were expressed in HeLa cells by using a vaccinia virus-T7 hybrid expression system. NS3 deltaC1459 and NS3F accumulated in the nucleus as well as in the cytoplasm, exhibiting a dot-like staining pattern. On the other hand, NS3 deltaNdeltaC was localized predominantly in the cytoplasm, suggesting the presence of a nuclear localization signal(s) in the amino-terminal sequence of NS3. NS4A, a viral cofactor for the NS3 protease, inhibited nuclear transport of NS3 deltaC1459 and NS3F, with the latter inhibited to a lesser extent than was the former. Interestingly, wild-type p53 tumor suppressor augmented nuclear localization of NS3 deltaC1459 and NS3F, whereas mutant-type p53 inhibited nuclear localization and augmented cytoplasmic localization of NS3 deltaC1459. However, subcellular localization of NS3 deltaNdeltaC was not affected by either type of p53. Wild-type p53-mediated nuclear accumulation of NS3 deltaC1459 and NS3F was inhibited partially, but not completely, by coexpressed NS4A, with NS3F again affected less prominently than was NS3 deltaC1459.

Wild-type, but not mutant-type, p53 enhances nuclear accumulation of the NS3 protein of hepatitis C virus.

By using vaccinia virus-T7 hybrid expression system, subcellular localization of the NS3 protein of hepatitis C virus was studied. Full-size NS3 (NS3F) and a carboxy-terminally truncated form (NS3 deltaC) were localized in the cytoplasm and the nucleus when expressed alone. However, NS3F and NS3 deltaC, but not amino- and carboxy-terminally truncated form (NS3 deltaN deltaC), were each co-localized with wild-type p53 almost exclusively in the nucleus upon co-expression. The wild-type p53-induced nuclear accumulation of NS3F was inhibited only partially by NS4A. When co-expressed with mutant-type p53, NS3F and NS3 deltaC were each co-localized with it exclusively in the cytoplasm. Taken together, the present results suggest that wild-type p53 enhances nuclear accumulation of NS3F and NS3 deltaC through the involvement of their amino-terminal sequences even in the presence of NS4A, and that mutant-type p53 inhibits their nuclear, and enhances their cytoplasmic, accumulation.

GB virus C/hepatitis G virus (GBV-C/HGV) infection in Chiang Mai, Thailand, and identification of variants on the basis of 5'-untranslated region sequences.

By using reverse transcription and PCR for NS3 and 5'-untranslated regions (5'UTR) of the viral genome, prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection in Chiang Mai, Thailand, was studied. High prevalence of GBV-C/HGV infection was observed among intravenous drug users (32%) and hemodialyzed patients (25%). The prevalence was also considerably high among patients with chronic liver disease, such as chronic hepatitis (9%), liver cirrhosis (12%) and hepatocellular carcinoma (10%). On the other hand, the prevalence among healthy blood donors (1%) was significantly lower than that of the above high-risk groups. GBV-C/HGV RNA positivity was significantly higher in individuals with antibodies against hepatitis C virus (24%) than in those without (5%). Phylogenetic analysis of the 5'UTR sequences classified Thai GBV-C/HGV isolates into three groups; (i) a group of isolates that are commonly found in the United States and Europe, (ii) a group of isolates that are commonly found in Asia, and (iii) a group of novel sequence variants.

Suppression of actinomycin D-induced apoptosis by the NS3 protein of hepatitis C virus.

The NS3 protein of hepatitis C virus is a multifunctional protein that is indispensable for virus replication. Little is known, however, about the possible effects of the NS3 on host cell function(s). In the present study, we demonstrated that NIH3T3 cells constitutively expressing a carboxy-terminally truncated NS3 (NS3DeltaC) were more resistant to actinomycin D-induced apoptosis than the control cells. We also observed that induction of p53 expression by actinomycin D treatment was weaker in the NS3DeltaC-expressing cells than in the control cells. However, induction of WAF1 expression by the same treatment was not different between the two groups. Taken together, our results suggest the possibility that expression of NS3DeltaC suppressed actinomycin D-induced apoptosis of NIH3T3 cells through at least partly, if not solely, a p53-dependent, WAF1-independent pathway.