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2.
J Virol ; 85(18): 9527-42, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21752919

ABSTRACT

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.


Subject(s)
Monkeypox virus/immunology , Monkeypox virus/pathogenicity , Mpox (monkeypox)/immunology , Mpox (monkeypox)/pathology , Viral Proteins/metabolism , Virulence Factors/metabolism , Adaptive Immunity , Animals , B-Lymphocytes/immunology , Blood/virology , DNA, Viral/chemistry , DNA, Viral/genetics , Disease Models, Animal , Female , Gene Deletion , Lung/virology , Macaca mulatta , Male , Molecular Sequence Data , Mpox (monkeypox)/virology , Primate Diseases/immunology , Primate Diseases/pathology , Primate Diseases/virology , Sequence Analysis, DNA , Skin/pathology , T-Lymphocytes/immunology , Viral Proteins/genetics , Virulence Factors/genetics
3.
Blood ; 112(10): 4227-34, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18757778

ABSTRACT

Rhesus macaque rhadinovirus (RRV) is closely related to Kaposi sarcoma-associated herpesvirus (KSHV) and is associated with the development of B-cell hyperplasia and persistent lymphadenopathy resembling multicentric Castleman disease in rhesus macaques (RMs) coinfected with simian immunodeficiency virus (SIV). Here we investigated whether RMs experimentally infected with SIV and RRV can develop other disease manifestations observed in HIV- and KSHV-infected patients. As reported earlier, inoculation of SIV-infected RMs with RRV results in persistent RRV infection, whereas immunocompetent animals infected with RRV exhibit viremia 2 weeks after infection, followed by a period of no virus detection until they are subsequently made immunodeficient by SIV infection. A subset of animals developed abnormal cellular proliferations characterized as extranodal lymphoma and a proliferative mesenchymal lesion. In situ hybridization and immunohistochemistry analysis indicate RRV is present in both malignancies, and DNA microarray analysis detected viral interleukin-6 (vIL-6) and viral FLICE-like inhibitory protein (vFLIP) transcripts. Reverse-transcriptase polymerase chain reaction analysis confirmed vIL-6 and vFLIP expression, and that of RRV open reading frames 72 and 73, homologs of KSHV open reading frames shown to be expressed in primary effusion lymphoma. These data support the utility of the RRV-/SIV-infected RM as an excellent animal model to investigate KSHV-like pathogenesis.


Subject(s)
Disease Models, Animal , HIV Infections/virology , HIV , Herpesviridae Infections/metabolism , Herpesvirus 8, Human/metabolism , Lymphoma, Non-Hodgkin/metabolism , Rhadinovirus/metabolism , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Immunodeficiency Virus , Tumor Virus Infections/metabolism , Animals , Castleman Disease/metabolism , Castleman Disease/virology , Gene Expression Regulation, Leukemic , Gene Expression Regulation, Viral , HIV Infections/metabolism , Herpesviridae Infections/virology , Humans , Lymphoma, Non-Hodgkin/virology , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/virology , Tumor Virus Infections/virology , Viral Proteins/biosynthesis
4.
J Virol ; 81(6): 2957-69, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17215283

ABSTRACT

Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV(17577)) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV(17577), producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.


Subject(s)
Chromosomes, Artificial, Bacterial , Herpesvirus 8, Human/genetics , Macaca mulatta/virology , Rhadinovirus/genetics , Sarcoma, Kaposi/etiology , Animals , Cells, Cultured , DNA, Viral/analysis , Fibroblasts/virology , Herpesvirus 8, Human/isolation & purification , Humans , Nucleic Acid Hybridization , Open Reading Frames , Polymerase Chain Reaction , Rhadinovirus/chemistry , Sarcoma, Kaposi/virology , Sequence Analysis, DNA
5.
Virology ; 344(2): 541-59, 2006 Jan 20.
Article in English | MEDLINE | ID: mdl-16199074

ABSTRACT

The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.


Subject(s)
Macaca nemestrina/virology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/pathogenicity , Viral Matrix Proteins/antagonists & inhibitors , Viral Matrix Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Amino Acid Sequence , Animals , CD4 Antigens/metabolism , Cell Line , Gene Expression Regulation, Viral , Human Immunodeficiency Virus Proteins , Lymphocytes/ultrastructure , Lymphocytes/virology , Molecular Sequence Data , Protein Engineering , Protein Structure, Tertiary , Protein Transport , RNA, Viral/blood , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Simian Immunodeficiency Virus/genetics , Viral Load , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics
6.
Virology ; 339(1): 56-69, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15975620

ABSTRACT

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.


Subject(s)
Acquired Immunodeficiency Syndrome/virology , HIV-1/genetics , Reassortant Viruses/physiology , Simian Immunodeficiency Virus/pathogenicity , Viral Regulatory and Accessory Proteins/physiology , Acquired Immunodeficiency Syndrome/immunology , Animals , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Cell Membrane/metabolism , Down-Regulation , Human Immunodeficiency Virus Proteins , Humans , Lymph Nodes/virology , Macaca nemestrina , Protein Structure, Tertiary/physiology , Reassortant Viruses/pathogenicity , Simian Immunodeficiency Virus/genetics , Virulence , Virus Replication
7.
Virology ; 313(2): 435-51, 2003 Sep 01.
Article in English | MEDLINE | ID: mdl-12954211

ABSTRACT

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV-1/pathogenicity , Protein Serine-Threonine Kinases/immunology , Reassortant Viruses/pathogenicity , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus , Viral Regulatory and Accessory Proteins/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , CD4 Lymphocyte Count , Casein Kinase II , Disease Models, Animal , Glycine/chemistry , Green Fluorescent Proteins , HIV-1/immunology , Human Immunodeficiency Virus Proteins , Luminescent Proteins/genetics , Macaca nemestrina , Molecular Sequence Data , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Reassortant Viruses/immunology , Sequence Alignment , Serine/chemistry , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Acquired Immunodeficiency Syndrome/virology , Viral Load , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics
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