The F gene of the Osaka-2 strain of measles virus derived from a case of subacute sclerosing panencephalitis is a major determinant of neurovirulence.

Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.

Remarkable similarity in genome nucleotide sequences between the Schwarz FF-8 and AIK-C measles virus vaccine strains and apparent nucleotide differences in the phosphoprotein gene.

The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains.

Contribution of matrix, fusion, hemagglutinin, and large protein genes of the CAM-70 measles virus vaccine strain to efficient growth in chicken embryonic fibroblasts.

Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution of each gene in the CAM-70 strain to efficient growth in chicken embryonic fibroblasts (CEF). We used a cloned MV IC323 based on the wild-type IC-B strain and generated a series of IC323s that possess one or more of the CAM-70 genes. Then, we examined the infection of CEF and CEF expressing human signaling lymphocyte activation molecule with the recombinant MVs. Our results demonstrated that MV needs to adapt to CEF at both the entry and postentry steps and that the CAM-70 matrix protein gene plays an important role in adaptation to CEF at the early stage of the virus replication cycle. The CAM-70 large protein gene was responsible for the efficient transcription and replication in CEF, and the CAM-70 hemagglutinin and fusion protein genes were responsible for efficient entry. Investigations focusing on these genes might elucidate unknown molecular mechanisms underlying the attenuation of MV.

Difference in production of infectious wild-type measles and vaccine viruses in monocyte-derived dendritic cells.

Macrophages (Mø) and dendritic cells (DC) are thought to be targets of measles virus (MeV) at the early stage of infection. We compared the growth of Edmonston-derived vaccine strains and fresh clinical isolates of MeV in monocytes, monocyte-derived granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced Mø (GM-Mø) and in monocyte-derived DC (Mo-DC). Neither vaccine strains nor fresh isolates thrived in monocytes and GM-Mø and no differences were evident among them. On the other hand, infectious virus production was robust in Mo-DC infected with fresh isolates, but below the limits of detection in those infected with vaccine strains. Although the vaccine strains infected Mo-DC and replicated comparably with the fresh isolates, they accumulated far less matrix (M) protein. This was attributed to a difference in the stability of M protein produced in Mo-DC between the strains. Impaired production of infectious viruses in DC may be one cause of vaccine strain attenuation.

Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation.

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell-cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell-cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.

Protection of macaques with diverse MHC genotypes against a heterologous SIV by vaccination with a deglycosylated live-attenuated SIV.

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates.

Reduced ability of hemagglutinin of the CAM-70 measles virus vaccine strain to use receptors CD46 and SLAM.

The CAM-70 measles virus (MV) vaccine strain is currently used for vaccination against measles. We examined the fusion-inducing ability of the CAM-70 hemagglutinin (H) protein and found that it was impaired in both CD46- and signaling lymphocyte activation molecule (SLAM)-expressing cells. We also generated recombinant MVs possessing H genes derived from the CAM-70 strain. The CAM-70 H protein impaired viral growth in both CD46- and SLAM-expressing cells. In peripheral blood lymphocytes (PBL) and monocyte-derived dendritic cells (Mo-DC), the CAM-70 strain did not grow efficiently. Infection with recombinant MVs revealed that impaired growth of the CAM-70 strain was attributed to the H gene only partly in PBL and largely in Mo-DC. Thus, impaired fusion-inducing ability of the H protein may be one of the underlying molecular mechanisms resulting in the attenuation of the CAM-70 strain.

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model.

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.

Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner.

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) has been implicated in inhibition of antiviral activity of IFN. While previous studies have suggested an interaction between NS5A and the double-stranded RNA-dependent protein kinase (PKR), the possibility still remains that interaction with another molecule(s) is involved in the NS5A-mediated inhibition of IFN. In the present study, we investigated a possible interaction between NS5A and 2',5'-oligoadenylate synthetase (2-5AS), another key molecule in antiviral activity. We observed that NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal portion of NS5A [aa 1-148; NS5A(1-148)] and two separate portions of 2-5AS (aa 52-104 and 184-275) being involved in the interaction. Single point mutations at residue 37 of NS5A affected the degree of the interaction with 2-5AS, with a Phe-to-Leu mutation (F37L) augmenting and a Phe-to-Asn mutation (F37N) diminishing it. Virus rescue assay revealed that the full-length NS5A (NS5A-F) and NS5A(1-148), the latter of which contains neither the IFN sensitivity-determining region (ISDR) nor the PKR-binding domain, significantly counteracted the antiviral activity of IFN. Introduction of a F37N mutation into NS5A(1-148) impaired the otherwise more significant IFN-inhibitory activity of NS5A(1-148). It was also found that the F37N mutation was highly disadvantageous for the replication of an HCV RNA replicon. Taken together, our results suggest the possibility that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.

c-MIR, a human E3 ubiquitin ligase, is a functional homolog of herpesvirus proteins MIR1 and MIR2 and has similar activity.

Kaposi's sarcoma associated-herpes virus encodes two proteins, MIR (modulator of immune recognition) 1 and 2, which are involved in the evasion of host immunity. MIR1 and 2 have been shown to function as an E3 ubiquitin ligase for immune recognition-related molecules (e.g. major histocompatibility complex class I, B7-2, and ICAM-1) through the BKS (bovine herpesvirus 4, Kaposi's sarcoma associated-herpes virus, and Swinepox virus) subclass of plant homeodomain (PHD) domain, termed the BKS-PHD domain. Here we show that the human genome also encodes a novel BKS-PHD domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 co-stimulatory molecule. This novel E3 ubiquitin ligase was designated as c-MIR (cellular MIR) based on its functional and structural similarity to MIR1 and 2. Forced expression of c-MIR induced specific down-regulation of B7-2 surface expression through ubiquitination, rapid endocytosis, and lysosomal degradation of the target molecule. This specific targeting was dependent upon the binding of c-MIR to B7-2. Replacing the BKS-PHD domain of MIR1 with the corresponding domain of c-MIR did not alter MIR1 function. The discovery of c-MIR, a novel E3 ubiquitin ligase, highlights the possibility that viral immune regulatory proteins originated in the host genome and presents unique functions of BKS-PHD domain-containing proteins in mammals.

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia.

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.

The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro.

Recombinant measles viruses (MV) in which the authentic glycoprotein genes encoding the fusion and the haemagglutinin (H) proteins of the Edmonston (ED) vaccine strains were swapped singly or doubly for the corresponding genes of a lymphotropic MV wild-type virus (strain WTF) were used previously to investigate MV tropism in cell lines in tissue culture. When these recombinants and their parental strains, the molecular ED-based clone (ED-tag) and WTF, were used to infect cotton rats, only viruses expressing the MV WTF H protein replicated in secondary lymphatic tissues and caused significant immunosuppression. In vitro, viruses containing the ED H protein revealed a tropism for human peripheral blood lymphocytes as documented by enhanced binding and virus production, whereas those containing the WTF H protein replicated well in monocyte-derived dendritic cells (Mo-DC). This did not correlate with more efficient binding of these viruses to DC, but with an enhancement of uptake, virus spread, accumulation of viral antigens and virus production. Thus, replacement of the ED H protein with WTF H protein was sufficient to confer the DC tropism of WTF to ED-tag in vitro. This study suggests that the MV H protein plays an important role in determining cell tropism to immune cells and this may play an important role in the induction of immunosuppression in vivo.