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PubMed1993

Development of recombinant vaccines against bluetongue.

Bluetongue virus is the aetiological agent of bluetongue, a disease of domestic and wild ruminants. Twenty-four serotypes are recognized. Novel subunit vaccines, that complement existing modified live polyvalent vaccines, are being developed. Serotype-specific viral neutralizing antibodies that are able to protect sheep against virulent homologous virus challenge can be induced by immunizing with the BTV outer capsid protein VP2 purified from virions or with VP2 expressed by baculovirus recombinants. Presentation of VP2 on virus-like particles, which assemble upon co-expression of the four major structural viral proteins (VP2, VP5, VP3 and VP7), improves the protective effect of VP2. Sheep immunized with core-like particles, comprised of VP3 and VP7, developed only limited clinical signs after virulent virus challenge, demonstrating that not only the outer capsid proteins, but also the core proteins are involved in protection against bluetongue.

Journal Article↗

PubMed1973

Direct evidence for messenger activity of influenza virion RNA.

In a cell-free system of Escherichia coli, RNA from influenza virus particles is translated into a polypeptide antigenically identical with the ribonucleoprotein and several more proteins, some of which correspond in size to viral structural components.

Animals↗

PubMed2003

Restriction of human immunodeficiency virus type 1 Rev function in murine A9 cells involves the Rev C-terminal domain.

The human immunodeficiency virus type 1 (HIV-1) Rev and human T-cell leukemia virus type 1 (HTLV-1) Rex proteins are essential for the expression of viral structural proteins and productive infection. Both contain a nuclear export signal (NES) in their C-terminal domain and a nuclear localization signal (NLS) in their N-terminal domain. The NES and NLS are necessary for shuttling between nucleus and cytoplasm and are therefore indispensable for the transport of unspliced and singly spliced viral transcripts. HIV-1 Rev function is restricted in A9 cells, a murine fibroblast cell line, whereas HTLV-1 Rex is functional in these cells. Immunofluorescence studies with RevGFP fusion protein demonstrate normal import and export of Rev in A9 cells. To ascertain which domains of Rev are necessary for the restriction of Rev function in A9 cells, we studied a chimeric construct in which the NES domain of Rev was exchanged with Rex C-terminal amino acids 79 to 95, the Rev1-79/Rex79-95 chimera, which restored Rev function in A9 cells. In addition, overexpression of a truncated Rev containing the Rev C-terminal domain in the presence of wild-type Rev, led to restoration of Rev function in A9 cells. These results suggest that the C-terminal domain of HIV-1 Rev plays an important role in restricting Rev function in murine cells.

Animals↗

PubMed1986

Effect of concanavalin A on early interactions of Sindbis virus with goose erythrocytes and BHK 21 cells.

The role of mannose containing molecules in Sindbis virus envelope glycoproteins and cell membrane receptors was investigated by means of concanavalin A (con A). Treatment of virus or BHK 21 cells with the lectin before infection reduced viral infectivity. Hemagglutination and hemolysis showed a different sensitivity to con A. Hemagglutination was not affected by the lectin and treatment of erythrocytes with con A produced an enhancement of agglutinability by the virus. Hemolytic activity was, on the contrary, strongly reduced by the lectin. The inhibition observed is due to an action on viral structures and not to an interaction with erythrocyte receptors because con A did not affect the hemolysis when preincubated with erythrocytes.

Animals↗

PubMed1995

Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

Amino Acid Sequence↗

PubMed2003

The abortive infection of Syrian hamster cells with human adenovirus type 12.

Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.

Adenoviruses, Human↗

PubMed2006

Inhibition of hepatitis C virus gene expression by small interfering RNAs using a tri-cistronic full-length viral replicon and a transient mouse model.

HCV is an ideal target for siRNA as its genome, a single-stranded RNA, is translated into a single viral polyprotein and replicated into negative-stranded RNA. In the present study, we monitored the effects of 36 different small interfering RNAs (siRNAs) transcribed from a plasmid-derived expression system on the luciferase activities expressed from a full-length HCV replicon, to identify potent siRNA target sites. Delivery of nine selected siRNA expression vectors into human hepatoma cells (Huh7) carrying a genomic HCV replicon resulted in a significant reduction in viral protein and RNA levels. Moreover, synthetic siRNAs directed to target sites (core, NS3, NS4A and NS4B coding regions) in the HCV genome efficiently suppressed viral replication in a dose-dependent manner. A transient mouse model system expressing viral structural proteins in the liver was constructed using the hydrodynamic transfection method to confirm in vivo anti-HCV activity of the selected siRNAs. A 21-nucleotide siRNA, which can hybridize to the HCV core coding region with a single G-U base pair, suppressed weakly transgene expression in mice. However, this anti-viral effect was enhanced upon substitution with a 27-mer duplex RNA. Our results will provide useful information about designing potent siRNAs against variable target sites.

Animals↗

PubMed1989

Infection of rats with bovine leukaemia virus: establishment of a virus-producing rat cell line.

Adult rats were infected with bovine leukaemia virus (BLV). Inoculated rats persistently produced antibodies directed against viral structural proteins. No major pathogenesis in infected rats was found during 2 years of observation. It was possible to recover the virus from rat spleen several months after infection. A cell line, R(BLV), was established from rat spleen; this contained integrated BLV provirus. R(BLV) cells kept for over 80 passages in vitro produced viral particles with the properties of BLV. Provirus reintegration and/or amplification occurred in R(BLV) cells. The cell line was found to be tumorigenic in rats, and the virus produced was immunogenic. R(BLV) cells represent the first described BLV-producing rat cell line. Proven persistent infection with BLV indirectly suggests that rats can serve as a reservoir of BLV in nature.

Animals↗

PubMed2001

Construction and in vitro properties of a series of attenuated simian immunodeficiency viruses with all accessory genes deleted.

We have generated simplified simian immunodeficiency virus (SIV) constructs lacking the nef, vpr, vpx, vif, tat, and rev genes (Delta6 viruses). To accomplish this, we began with an infectious molecular clone of SIV, i.e. SIVmac239, and replaced the deleted segments with three alternate elements: (i) a constitutive transport element (CTE) derived from simian retrovirus type 1 to replace the Rev/Rev-responsive element (RRE) posttranscriptional regulation system, (ii) a chimeric SIV long terminal repeat (LTR) containing a cytomegalovirus (CMV) promoter to augment transcription and virus production, and (iii) an internal ribosome entry site (IRES) upstream of the env gene to ensure expression of envelope proteins. This simplified construct (Delta6CCI) efficiently produced all viral structural proteins, and mature virions possessed morphology typical of wild-type virus. It was also observed that deletion of the six accessory genes dramatically affected both the specificity and efficiency of packaging of SIV genomic RNA into virions. However, the presence of both the CTE and the chimeric CMV promoter increased the specificity of viral genomic RNA packaging, while the presence of the IRES augmented packaging efficiency. The Delta6CCI virus was extremely attenuated in replication capacity yet retained infectiousness for CEMx174 and MT4 cells. We also generated constructs that retained either the rev gene or both the rev and vif genes and showed that these viruses, when complemented by the CMV promoter, i.e., Delta5-CMV and Delta4-CMV, were able to replicate in MT4 cells with moderate and high-level efficiency, respectively. Long-term culture of each of these constructs over 6 months revealed no potential for reversion. We hope to shortly evaluate these simplified constructs in rhesus macaques to determine their long-term safety as well as ability to induce protective immune responsiveness as proviral DNA vaccines.

Animals↗

PubMed1985

Nucleotide sequence of a human cytomegalovirus DNA fragment encoding a 67-kilodalton phosphorylated viral protein.

Previously, we demonstrated that a DNA fragment from the L-unique region at 0.37 to 0.39 map units of human cytomegalovirus DNA codes for a 67-kilodalton phosphorylated tegument protein. (Davis et al., J. Virol. 52:129-135, 1984). We have determined the nucleotide sequence of this DNA, transcription initiation and termination, and putative promoter region. We also compared the properties of the protein product predicted from the DNA sequence with the properties of the protein isolated by immunoabsorbent column chromatography. This viral structural protein may have important functions, such as protein kinase activity, DNA binding, and possible transcriptional activation of immediate-early genes.

Amino Acid Sequence↗

PubMed1982

Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus.

Several functions of the retrovirus genome involve structural features in the vicinity of its 5' terminus. In an effort to further elucidate the relationship between structure and function in retrovirus RNA, we have determined the sequence of the first 1,010 nucleotides at the 5' end of the genome of Rous sarcoma virus by using the Maxam-Gilbert method to sequence suitable domains in cloned Rous sarcoma virus DNA. The results (i) locate the initiation codon for the gag gene of Rous sarcoma virus 372 nucleotides from the 5' end of viral RNA; (ii) demonstrate that this codon is preceded by three methionine codons that are apparently not used in translation; (iii) sustain previous conclusions that the principal site to which ribosomes bind on the Rous sarcoma virus genome in vitro does not contain the initiation codon for gag; (iv) permit deduction of the amino acid sequence of a viral structural protein, p19; (v) confirm the amino-terminal sequence of Pr76gag; and (vi) substantiate the identification of a splice donor site described in the accompanying manuscript (Hackett et al., J. Virol., 41:527-534, 1982).

Avian Sarcoma Viruses↗

PubMed1994

Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes.

Primary cytomegalovirus (CMV) infection and reactivation of persistent CMV are associated with significant morbidity and mortality in immunocompromised individuals. Although recovery from CMV disease is correlated with the development of CMV-specific cytotoxic T lymphocytes (CTL), the major viral target antigens to which the response is directed are ill-defined, though they may comprise viral structural elements. We now identify the CMV matrix protein pp65 as a significant target antigen for CD8+ class I major histocompatibility complex (MHC)-restricted CMV-specific CTL derived from the peripheral blood of four of five latently infected individuals. CMV-specific CTL recognition of pp65 on target cells occurs prior to the onset of viral gene expression and persists throughout the duration of the replicative cycle. Recognition in the absence of viral gene expression suggests that abundant viral protein enters the normal trafficking pathway upon viral penetration and is readily made available to MHC molecules for presentation at the cell surface. Thus pp65 specific CTL may represent an important effector population for early control and limitation of CMV infection and disease. The observation that CMV-specific CTL can be induced in vitro using peptide fragments derived from pp65 supports the future use and manipulation of this and similar effector populations in a clinical setting.

Antigens, Viral↗

PubMed1976

Virus-cell interaction in oligodendroglia, astroglia and phagocyte in progressive multifocal leukoencephalopathy. An electron microscopic study.

A 46-year-old female, with an 11 year history of malignant lymphoreticular disease, developed a neurological illness clinically manifested by a focal mass lesion in the left frontal lobe. In biopsied tissue, immunofluorescence study revealed the presence of JC antigen in the glial cells. Histologically, the lesion was characteristic of PML consisting of PML consisting of focal necrosis in the subcortical white matter, numerous fat laden macrophages and marked hypertrophy of oligodendrocytes and astrocytes. By electron microscopy, hypertrophic astrocytes contained intranuclear viral particles consistent with papova virions and aggregates of intracytoplasmic viral particles consisting of a single to several virions tightly surrounded by a single membrane. The membrane appeared to have been derived from that of the cellular vesicles. Fusion of the virus-associated membrane to the astroglial plasmalemma occurred when the virions appeared to shift towards extracellular space. The virioncontaining astrocytes showed cytoplasmic "fibrillar hypertrophy" similar to the characteristic gigantic astroglias of PML. This fact would provide an additional evidence that these gigantic cells, although lacking identifiable viral structures, were the result of anaplastic transformation by JC virus. Many virus-bearing astroglias were noted to be in the early stage of cellular necrosis, of "edematous degeneration". This further indicates that the JC virus is capable of inducing both lytic and abortive astroglial infections. Many oligodendroglias were hypertrophic due to the presence of intranuclear viral particles and markedly increased numbers of microtubules and free ribosomes in the cytoplasm. Membrane-bound intracytoplasmic viral particles were also noted in the oligodendroglias. Some fat laden macrophages contained large intracytoplasmic viral bodies, presumably originating from phagocytized virus-bearing cells.

Astrocytes↗

PubMed1990

Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis.

A 'blind' recombinant immunoscreening approach, of general application to studies of infectious diseases, was used to clone and identify the genome of the previously uncharacterized non-A, non-B hepatitis (NANB) virus. This agent is a positive-stranded RNA virus that appears to be distantly related to the flaviviridae family. A recombinant viral antigen (C100-3) was used to develop a capture assay for circulating antibody. Data obtained using this assay indicate that this agent, termed the hepatitis C virus (HCV), is the major cause of post-transfusion, community-acquired and cryptogenic, NANB. Anti-C100-3 antibody appears to be directed towards dominant, non-structural viral epitopes. It is a non-neutralising antibody that develops generally late in infection and is a particularly good marker of chronic, persistent viraemia. Many asymptomatic but infectious blood donors can now be detected using this antibody assay. HCV is associated with the development of hepatocellular carcinoma and possibly, other liver diseases.

Amino Acid Sequence↗

PubMed2003

Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3' untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication.

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A Renilla luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3' untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5' region of the capsid gene (5'CS) abolished RNA replication. Restoration of the 5'CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5'CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.

3' Untranslated Regions↗

PubMed2000

HIV-1 infected mononuclear phagocyte secretory products affect neuronal physiology leading to cellular demise: relevance for HIV-1-associated dementia.

Viral and cellular products from HIV-1-infected and/or immune competent mononuclear phagocytes (MP) (brain macrophages and microglia) affect neuronal function during HIV-1-associated dementia (HAD). Neurotoxic MP factors include, but are not limited to, pro-inflammatory cytokines, chemokines, platelet activating factor, arachidonic acid and its metabolites, nitric oxide, progeny virions and viral structural and regulatory proteins. The mechanisms for immune-mediated neural injury in HAD, only now, are being unraveled. In this regard, we reviewed the current knowledge of how postmitotic neurons, which can neither divide nor be replaced, are damaged by MP secretory activities. Linking neuronal function with brain MP activation was made possible by placing viral and/or immune products onto neurons and measuring cell signaling events or through ex vivo electrophysiological tests on MP-treated brain slices. Such linkages are shown, in this report, by select demonstrations of MP factors which cause neuronal dysfunction in HAD.

AIDS Dementia Complex↗

PubMed1997

Intracellular viral localization in murine coxsackievirus-B3 myocarditis. Ultrastructural study by electron microscopic in situ hybridization.

Group B Coxsackieviruses are a common cause of myocarditis. To detect the viral genome and its localization in the myocardium, we examined C3H/He mice with Coxsackievirus B3 (CVB3) myocarditis on days 5, 8, and 14 after inoculation by the reverse transcriptase polymerase chain reaction and by in situ hybridization. Sense and antisense CVB3 RNA were detected in the myocardium of all mice up to day 14 by reverse transcriptase polymerase chain reaction. Light microscopic in situ hybridization with a cDNA probe for CVB3 showed clusters of positive signals in the areas of myocardial necrosis and cell infiltration. With electron microscopic in situ hybridization, CVB3 RNA was detected in the cytoplasm of cardiocytes, between the myofibrils, near the mitochondria, and in tubular or vesicular structures. Viral RNA was also detected in necrotic debris, in the cytoplasm of macrophages, and in the cytoplasm of interstitial fibroblasts. These findings suggest that CVB3 RNA is replicated in the cytoplasm of cardiocytes, transferred into tubular or vesicular structures, released into the interstitium, and phagocytosed by macrophages. Some positive signals were also detected in the cytoplasm of cardiocytes showing close contact with infiltrating lymphocytes, suggesting that the lymphocytes recognized virus-infected cardiocytes and caused cell-mediated immune cardiocyte damage.

Animals↗

PubMed1971

Temperature-sensitive mutants of vesicular stomatitis virus: synthesis of virus-specific proteins.

Viral proteins synthesized in L cells infected with temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus at permissive (31 C) and nonpermissive (39 C) temperatures were compared by polyacrylamide gel electrophoresis. Mutant ts 5, deficient in synthesis of viral ribonucleic acid (RNA(-)), failed to synthesize any of the five identifiable viral proteins at 39 C. Each of three RNA(+) mutants, representing three separate complementation groups, showed distinctive patterns of viral protein synthesis at nonpermissive temperature. Equivalent amounts of (3)H-amino acids were incorporated into the five viral proteins made in cells infected with RNA(+) mutant ts 45 at 31 and 39 C. Complete virions of ts 45 could be identified by electron microscopy of infected cells incubated at the nonpermissive temperature; the defect in ts 45 appeared to be due in part to greater thermolability of virions as compared with the wild-type. RNA(+) mutant ts 23 was deficient in synthesis of viral envelope protein S and failed to make detectable virions at the nonpermissive temperature. Infection of cells at 39 C with the third RNA(+) mutant, ts 52, resulted in synthesis of all five viral proteins, but the peak of radioactivity representing the viral membrane glycoprotein migrated more rapidly on gels than coelectrophoresed authentic virion (14)C-glycoprotein or viral (3)H-glycoprotein extracted from cells infected at 31 C. These data and results of experiments on incorporation of radioactive glucosamine suggest that the primary defect in mutant ts 52 at nonpermissive temperature is failure of glycosylation of the viral glycoprotein. The viral structural proteins made in cells infected with ts 52 at the nonpermissive temperature did not assemble into sedimentable components as they did at permissive temperature; this observation indicates failure of insertion of the nonglycosylated protein (G') into cell membrane. In support of this hypothesis was the finding that antiviral-antiferritin hybrid antibody did not detect VS viral antigen on the plasma membrane of L cells infected at 39 C with ts 52. In contrast, VS viral antigen localized in plasma membrane of L cells infected at 39 C with mutants ts 23 and ts 45 was readily detected by electron microscopy and fluorescence microscopy.

Amino Acids↗