Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis.

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.     

Change in host cell tropism associated with in vitro replication of equine infectious anemia virus.

Similar to other human and animal lentiviruses, equine infectious anemia virus (EIAV) is detectable in vivo in cells of the monocyte-macrophage lineage. Owing to their short-lived nature, horse peripheral blood macrophage cultures (HMC) are rarely used for in vitro propagation of EIAV, and equine dermal (ED) or kidney cell cultures, which can be repeatedly passed in vitro, are used in most studies. However, wild-type isolates of EIAV will not grow in these cell types without extensive adaptation, a process which may attenuate viral virulence. To better define the effect of host cell tropism on the virulence and pathogenesis of EIAV, we studied a field isolate of EIAV during in vitro adaptation to growth in an ED cell line. Interestingly, as the virus adapted to growth in ED cells, there was a corresponding decrease in infectivity for HMC, and the final ED-adapted isolate was more than 100-fold more infectious for ED cells than for HMC. In vivo studies indicated that the ED-adapted isolate was able to replicate in experimentally infected horses, although no clinical signs of EIA were observed. Thus, selection for in vitro replication on ED cells correlated with a loss of EIAV tropism for HMC in vitro and was associated with avirulence in vivo.     

Differential cell tropism of feline immunodeficiency virus molecular clones in vivo

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4(+) and CD8(+) lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4(+) lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4(+) and CD8(+) lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.     

An in vitro DNA virus for in vitro protein evolution.

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.     

Host modification of Sindbis virus sialic acid content influences alternative complement pathway activation and virus clearance

Previous studies have shown that Sindbis virus, an enveloped alphavirus of the togavirus group, activates the alternative complement pathway in the absence of detectable antiviral immunoglobulin. The present studies examined the role of the host-determined sialic acid content of Sindbis virus on activation of the alternative complement pathway. Purified Sindbis virus grown in baby hamster kidney (BHK-SV) and in mosquito (MOSQ-SV) cells yielded virus with 10.2 and less than 2.0 nmol sialic acid/mg viral protein, respectively. Sindbis virus deficient in sialic acid (2.0 nmol sialic/mg) was also produced by treating the BHK-SV with neuraminidase (NANase-SV). When MOSQ-SV or NANase-SV was incubated in either C4DGPS or C2DHS, each consumed significantly more C3 than did BHK-SV, indicating that the ability of Sindbis virus to activate the alternative pathway is inversely related to its sialic acid content. Studies in vivo showed that virus deficient in sialic acid (MOSQ-SV) was cleared from the blood of mice much more efficiently than was virus rich in sialic acid (BHK-SV), after i.v. inoculation. Furthermore, when animals were depleted of C3 through C9 by cobra venom factor (CoVF) treatment, no differences in the clearance of high and low sialic acid-containing viruses were observed. Thus both the activation in vitro and complement-dependent clearance in vivo are significantly affected by the host-determined sialic acid content of Sindbis virus.     

Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors

BACKGROUND: Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS: The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS: In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS: This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.     

Application of an immunoassay to the study of yeast malate dehydrogenase inactivation.

In derepressed yeast cells the cytoplasmic malate dehydrogenase activity disappears after addition of glucose to the culture medium. Using specific antisera, it seemed possible to isolate an inactive enzyme protein if the inactivation resulted from an allosteric inhibition or from a chemical modification. The present studies show that after the inactivation an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of the inactivation in vivo and in vitro this result supports a proteolytic mechanism of enzyme inactivation.     

Characterization of early pathogenic effects after experimental infection of calves with bovine immunodeficiency-like virus.

The early pathogenic effects of bovine immunodeficiency-like virus (BIV) were studied in calves experimentally inoculated with BIV. All animals inoculated with BIV R29-infected cells seroconverted by 6 weeks postinoculation, and BIV was recoverable from each animal at 2 weeks postinoculation. However, levels of BIV replication in vivo appeared to be low. In situ hybridization studies indicated that during peak periods of viral replication in vivo, less than 0.03% of peripheral blood mononuclear cells were expressing detectable levels of viral RNA. Moreover, the levels of viral RNA in these cells in vivo were less than 1/10 the levels observed in persistently infected cells in vitro. BIV-inoculated calves had significantly higher numbers of circulating lymphocytes, and follicular hyperplasia was observed in lymph nodes, hemal nodes, and spleen. The histopathological changes observed in BIV-infected calves were similar to changes found early after infection with the immunosuppressive lentiviruses, including human immunodeficiency virus type 1.     

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

In Vivo Monocyte Tropism of Pathogenic Feline Immunodeficiency Viruses

Virus-infected monocytes rarely are detected in the bloodstreams of animals or people infected with immunodeficiency-inducing lentiviruses, yet tissue macrophages are thought to be a major reservoir of virus-infected cells in vivo. We have identified feline immunodeficiency virus (FIV) clinical isolates that are pathogenic in cats and readily transmitted vertically. We report here that five of these FIV isolates are highly monocytotropic in vivo. However, while FIV-infected monocytes were numerous in the blood of experimentally infected cats, viral antigen was not detectable in freshly isolated cells. Only after a short-term (at least 12-h) in vitro monocyte culture were FIV antigens detectable (by immunocytochemical analysis or enzyme-linked immunosorbent assay). In vitro experiments suggested that monocyte adherence provided an important trigger for virus antigen expression. In the blood of cats infected with a prototype monocytotropic isolate (FIV subtype B strain 2542), infected monocytes appeared within 2 weeks, correlating with high blood mononuclear-cell-associated viral titers and CD4 cell depletion. By contrast, infected monocytes could not be detected in the blood of cats infected with a less pathogenic FIV strain (FIV subtype A strain Petaluma). We concluded that some strains of FIV are monocytotropic in vivo. Moreover, this property may relate to virus virulence, vertical transmission, and infection of tissue macrophages.     

Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.     

EB病毒转化Hu－TLC－SCID小鼠脾细胞的实验研究

利用ＥＢ病毒转化可产生较高水平人ＩｇＧ和特异性抗２型登革病毒人抗体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞,通过免疫组化、免疫荧光和ＰＣＲ法检测转化细胞的人Ｂ细胞表面标志、ＥＢ病毒抗原和ＥＢ病毒基因。结果表明,被转化的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞能继续产生抗２型登革病毒的特异性人抗体,并具有人Ｂ细胞的、ＳｍＩｇＧ标志及ＥＢ病毒潜伏膜蛋白－１（ＬＭＰ－１）基因,可表达ＬＭＰ－１和ＥＢ病毒核抗原（ＥＢＮＡ）。     

Posttranslational modifications as a cause of gonadal polypeptide diversity in the rat

The testicular and ovarian protein patterns of prepubertal rats were studied by two-dimensional gel electrophoresis after both in vivo and in vitro translation. Analysis of the approximately 750 polypeptide spots detectable in gonadal extracts (in vivo translated polypeptides) revealed 14 testis-specific, and 2 ovary-specific spots. Conversely, only 4 testis- and 2 ovary-specific polypeptide spots (of a total of about 600) were detected after in vitro translation of the respective polyadenylated RNAs using the reticulocyte lysate system. Simultaneous in vitro translation plus microsome-mediated cotranslational modification increased the number (of testicular spots to 8, and of ovarian spots to 3 (of a total of 600). The polypeptides synthesized in vitro were well comparable with respect to molecular weight and isoelectric point to those synthesized in vivo. Therefore, testicular polypeptide diversity is mainly the result of co/posttranslational modifications. Many of these proteins appeared to be glycosylated as indicated by their specific binding to Concanavalin A. The system used demonstrates that co/posttranslational modifications play a role in the establishment of polypeptide and thus organ diversity.     

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.     

Immune activation and viral burden in acute disease induced by simian immunodeficiency virus SIVsmmPBj14: correlation between in vitro and in vivo events.

The simian immunodeficiency virus SIVsmmPBj14 (SIV-PBj14) is an atypical lentivirus that causes acute disease and death in pig-tailed macaques and in vitro replicates efficiently in resting macaque lymphocytes and activates and induces proliferation of lymphocytes. The present study was conducted to test the hypothesis that production of large quantities of SIV-PBj14 induces widespread immune activation and elaboration of cytokines which lead directly to the death of infected pig-tailed macaques. Following intravenous inoculation of pig-tailed macaques with SIV-PBj14, acute disease developed and was characterized by high levels of plasma viremia, p27gag antigenemia, tumor necrosis factor alpha, and interleukin-6 (IL-6). All animals died within 10 days of infection, at which time some animals had as many as 100% CD4+ cells in the periphery and lymphoid tissues infected. During the last few days before death, titers of infectious virus in blood increased as much as 10(5)-fold. By using dual-label immunofluorescence assays for detection of cell surface activation markers, both CD4+ and CD8+ lymphocytes were shown to express the IL-2 and transferrin receptors following either in vivo or in vitro infection with SIV-PBj14. Furthermore, in vitro infection of quiescent macaque lymphocytes by SIV-PBj14 was accompanied by proliferation of both CD4+ and CD8+ lymphocyte subsets, as measured by incorporation of [3H]thymidine. Increases in numbers of activated lymphocytes and levels of proinflammatory cytokines in plasma coincided with increased amounts of detectable virus in vivo. Clinical signs of disease and pathologic findings were most consistent with death from a shock-like syndrome, in which acute-phase inflammatory cytokines are known to play a major role. Tumor necrosis factor alpha, IL-2, and IL-6 were detected in some cultures infected with SIV-PBj14, but this finding was not consistent. When cytokines were detected, their concentrations were essentially no different from those found in control cultures infected with SIVsmm9, a prototypic strain from which SIV-PBj14 was derived. The in vivo results suggest a synergistic cycle of activation of lymphocytes and monocytes, elaboration of cytokines, and virus production that accelerates uncontrolled and culminates in death. The observed correlations between in vivo and in vitro activation events following SIV-PBj14 infection validate the use of in vitro studies to clarify lentivirus-lymphocyte interactions that may contribute to the virulence of SIV-PBj14.     

First snapshots of the HIV-1 RNA structure in infected cells and in virions

With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.     

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.     

Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element.

Expression of the structural proteins of human T-cell leukemia virus type I is dependent upon the interaction of the viral Rex trans activator with its highly structured cis-acting RNA target sequence, the 254-nucleotide Rex-response element. Nucleotides critical for Rex binding in vitro have been mapped by modification interference analysis to a discrete 12-nucleotide RNA sequence that is predicted to form a stem-bulge-stem structure. This minimal RNA binding site was sufficient to mediate specific Rex binding in vitro when analyzed in the context of a short RNA probe. The critical importance of this short RNA sequence in mediating Rex function in vivo is supported by its complete conservation among all primate T-cell leukemia virus isolates.     

Host-Range Restrictions of Murine Leukemia Viruses in Mouse Embryo Cell Cultures

Murine leukemia virus strains fall into three categories with respect to their ability to propagate in cells of National Institutes of Health (NIH) Swiss and BALB/c mouse embryos. Cultures of NIH cells are 100- to 1,000-fold more sensitive to "N-tropic" strains than BALB/c cell cultures, but are 30- to 100-fold less sensitive to "B-tropic" strains. Some virus strains (dually tropic or "NB-tropic") propagate equally well in both cells. M-MSV pseudotypes show the host-range characteristics of the virus supplying the envelope, both in vitro and in vivo. The host-range characteristics appear to be genetically determined and could not be explained by host-induced modification or virus mixtures. There was no correlation between host range and Gross-AKR or FMR serotype.     

New mammalian transforming retrovirus: demonstration of a polyprotein gene product.

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.