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PubMed2001

Targeted gene transfer to lymphocytes using murine leukaemia virus vectors pseudotyped with spleen necrosis virus envelope proteins.

In contrast to murine leukaemia virus (MLV)-derived vector systems, vector particles derived from the avian spleen necrosis virus (SNV) have been successfully targeted to subsets of human cells by envelope modification with antibody fragments (scFv). However, an in vivo application of the SNV vector system in gene transfer protocols is hampered by its lack of resistance against human complement. To overcome this limitation we established pseudotyping of MLV vector particles produced in human packaging cell lines with the SNV envelope (Env) protein. Three variants of SNV Env proteins differing in the length of their cytoplasmic domains were all efficiently incorporated into MLV core particles. These pseudotype particles infected the SNV permissive cell line D17 at titers of up to 10(5) IU/ml. A stable packaging cell line (MS4) of human origin released MLV(SNV) pseudotype vectors that were resistant against human complement inactivation. To redirect their tropism to human T cells, MS4 cells were transfected with the expression gene encoding the scFv 7A5 in fusion with the transmembrane domain (TM) of the SNV Env protein, previously shown to retarget SNV vector particles to human lymphocytes. MLV(SNV-7A5)-vector particles released from these cells were selectively infectious for human T cell lines. The data provide a proof of principle for targeting MLV-derived vectors to subpopulations of human cells through pseudotyping with SNV targeting envelopes.

Animals↗
PubMed2004

Genetic relatedness of deer mice (Peromyscus maniculatus) infected with Sin Nombre virus.

The deer mouse (Peromyscus maniculatus) is the primary rodent host of Sin Nombre virus (SNV), the principal etiologic agent of hantavirus pulmonary syndrome in the United States. Many characteristics of SNV infections of deer mice are unknown. To better understand the transmission mechanisms of SNV in deer mice, we conducted mark-recapture sampling and genetic analyses to study deer mouse population density and genetic relatedness from 2001 to 2002. We genotyped each deer mouse at 10 microsatellite loci to estimate relatedness among SNV-infected and SNV-uninfected groups, demographic categories, and individuals. Estimates of average overall population densities ranged from 1.15 to 14.95 deer mice/ha. Estimates of average population densities of SNV-infected deer mice ranged from 0 to 1.55 deer mice/ha. When evaluated as groups at one of two study sites, SNV-infected deer mice were more related to one another, on average, than they were to their uninfected cohorts. Pairwise coefficients of relatedness among individuals indicated that several deer mice infected with SNV were closely related. Most infected mice were males. These results are consistent with the hypothesis that transmission of SNV often is associated with breeding activities and aggression among males during the breeding season and suggest that post-birth behavioral associations among adults and juveniles may be a factor in the transmission of SNV.

Animals↗
PubMed1999

Temporal artery biopsy: a diagnostic tool for systemic necrotizing vasculitis. French Vasculitis Study Group.

OBJECTIVE: To describe the clinical, biologic, and histologic features of temporal artery biopsy (TAB)-localized systemic necrotizing vasculitides (SNV), and to assess their frequency among elderly patients undergoing TAB for suspected giant cell (temporal) arteritis (GCA). METHODS: The frequency of a TAB localization of SNV was prospectively assessed in a multicenter study of elderly patients undergoing TAB for suspected GCA. All patients with SNV fulfilling the American College of Rheumatology criteria for a specific vasculitic syndrome and with evidence of vasculitis on TAB were included in a retrospective, descriptive study. RESULTS: SNV was diagnosed based on the TAB in 1.4% of the patients with suspected GCA and in 4.5% of the positive (inflamed) TAB specimens. We retrospectively selected 27 patients (18 female, 9 male; mean +/- SD age 62+/-15 years, range 22-79 years) with SNV and TAB-localized vasculitis. Only 2 of these patients were known to have SNV before TAB localization. Twenty-two patients (81%) had cephalic symptoms, including jaw claudication in 33%, clinically abnormal temporal arteries in 33%, and neuro-ophthalmologic symptoms in 11%. All patients had systemic symptoms suggestive of SNV and histologically proven NV in the TAB specimens (70%) or elsewhere in other biopsy sites (74%). Abnormal biologic results suggestive of SNV were present in 17 patients (63%). For 4 patients, the TAB-documented involvement led to initial misdiagnoses of GCA, and systemic manifestations that developed under steroid therapy revealed the correct diagnosis. The final diagnoses of the patients were polyarteritis nodosa (PAN) (n = 11), Churg-Strauss syndrome (n = 6), micropoly-angiitis (n = 3), Wegener's granulomatosis (n = 3), hepatitis B virus-related PAN (n = 2), hepatitis C virus-related cryoglobulinemic vasculitis (n = 1), and rheumatoid vasculitis (n = 1). CONCLUSION: TAB-localized SNV presents a major diagnostic dilemma because it can mimic GCA. Careful analysis of clinical, biologic, and histologic data should lead to the correct diagnosis and help guide the clinician's choice of appropriate therapy.

Adrenal Cortex Hormones↗
PubMed2004

Identification and analysis for cross-reactivity among hantaviruses of H-2b-restricted cytotoxic T-lymphocyte epitopes in Sin Nombre virus nucleocapsid protein.

Sin Nombre virus (SNV) causes hantavirus pulmonary syndrome (HPS), with a high rate of mortality in humans who are infected by the transmission of virus from the natural rodent host. In humans, cytotoxic T lymphocytes (CTL) specific for SNV appear to play an important role in the pathogenicity of HPS. There is a correlation between the frequencies of SNV-specific CTLs and the severity of HPS disease. In order to create a mouse model to study the role of SNV-specific T cells in vivo, T cell responses to SNV nucleocapsid (N) protein in B6.PL Thy1(a)/Cy mice (H-2(b)) immunized with plasmid DNA or recombinant vaccinia virus expressing SNV N protein were examined. Four peptides, NC94-101, NC175-189, NC217-231 and NC331-345, were recognized by CD8(+) T cells in CTL and ELISPOT assays in SNV N-immunized mice. Interestingly, two of these epitopes are located in the central region of the SNV N protein, where several human CD8(+) T-cell epitopes have been defined in Puumala virus and SNV. CTL lines specific for these four epitopes were cross-reactive to corresponding Puumala virus peptides, but only one of them was cross-reactive to Hantaan virus peptides. These results will enable the analysis of the roles of CTL in immunopathology of HPS in experimental mouse models of HPS.

Amino Acid Sequence↗
PubMed1997

Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis.

To develop a rapid antibody test for Sin Nombre hantavirus (SNV) infection for diagnosis of hantavirus pulmonary syndrome (HPS) in field settings where advanced instrumentation is not available, a strip immunoblot assay bearing four immobilized antigens for SNV and a recombinant nucleocapsid protein antigen of Seoul hantavirus (SEOV) was prepared. The SNV antigens included a full-length recombinant-expressed nucleocapsid (N) protein (rN), a recombinant-expressed G1 protein (residues 35 to 117), and synthetic peptides derived from N (residues 17 to 59) and G1 (residues 55 to 88). On the basis of the observed reactivities of hantavirus-infected patient and control sera, we determined that a positive assay requires reactivity with SNV or SEOV rN antigen and at least one other antigen. Isolated reactivity to either viral rN antigen is indeterminate, and any pattern of reactivity that does not include reactivity to an rN antigen is considered indeterminate but is unlikely to represent hantavirus infection. Fifty-eight of 59 samples from patients with acute SNV-associated HPS were positive according to these criteria, and one was initially indeterminate. Four of four samples from patients with HPS due to other hantaviruses were positive, as were most samples from patients with SEOV and Puumala virus infections. Of 192 control serum samples, 2 (1%) were positive and 2 were indeterminate. Acute SNV infection was distinguishable from remote SNV infection or infection with hantaviruses other than SNV by the presence of G1 peptide antigen reactivities in the former. The strip immunoblot assay shows promise for the detection of SNV antibodies early in the course of HPS.

Antibodies, Viral↗
PubMed1987

Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells.

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.

Animals↗
PubMed1991

Spleen necrosis virus, an avian retrovirus, can infect primate cells.

Spleen necrosis virus (SNV) is an avian retrovirus that can infect some mammalian cells such as dog cells as well as all avian cells tested to date. We were interested in testing whether SNV could also infect primate cells. For these experiments, we used HeLa and COS-7 cells. Initially, we determined whether the SNV long terminal repeat promoter was functional in HeLa and COS-7 cells. In transient transfection assays, the SNV promoter efficiently directed chloramphenicol acetyltransferase gene expression in both HeLa and COS-7 cells. Using SNV- and murine leukemia virus-derived retroviral vectors containing the neomycin phosphotransferase gene, we found that SNV established a provirus in HeLa and COS-7 cells as efficiently as did an amphotropic murine leukemia virus, as judged by the number of G418-resistant HeLa and COS-7 cell colonies obtained after infection and selection. Although SNV formed a provirus in both HeLa and COS-7 cells, productive infection of these cells was not obtained with use of replication-competent SNV. These results suggest that SNV can infect, form a provirus, and stably express a transduced gene in primate cells, but there is a posttranscriptional block to its replication in these cells.

Animals↗
PubMed1999

The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production.

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20, 000-fold by the 5' SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3' LTR and 3' untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5' RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5' RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.

Animals↗
PubMed2004

Cross-packaging of human immunodeficiency virus type 1 vector RNA by spleen necrosis virus proteins: construction of a new generation of spleen necrosis virus-derived retroviral vectors.

The ability of the nonlentiviral retrovirus spleen necrosis virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retrovirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.

Amino Acid Sequence↗
PubMed2002

Assessment of ecologic and biologic factors leading to hantavirus pulmonary syndrome, Colorado, U.S.A.

AIM: To understand the ecologic parameters of Sin Nombre virus (SNV; family Bunyaviridae, genus Hantavirus) infections in the deer mouse (Peromyscus maniculatus), environmental variables impacting the rodent populations, and the conditions under which SNV is amplified. This may help us understand the antecedents of human risk for developing hantavirus pulmonary syndrome (HPS) as a consequence of SNV infection. METHOD: Each 6 weeks, we trapped, measured, tagged, bled, and released rodents at three widely spaced sites in Colorado, USA: Fort Lewis (1994-2001), Molina (1994-2001), and Pinyon Canyon Maneuver Site (1995-2001). The ELISA method was used to test rodent blood samples for IgG antibody to SNV antigen. RESULTS: Where rodent species richness was high, the prevalence of infection of deer mice (as determined by the presence of antibody) with SNV was low, and vice versa. There was a higher prevalence of antibody to SNV in male than in female rodents, and seasonal differences were observed in acquisition of SNV between male and female deer mice. Long-lived infected deer mice served as transseasonal, over-winter reservoirs for the virus, providing the mechanism for its survival. CONCLUSION: Prevalence of rodent infection appears to be associated with fluctuations in deer mouse populations and, indirectly, with timing and amount of precipitation and the resulting biologic events (a trophic cascade). Together with information regarding transseasonal maintenance of SNV, seasonal differences in acquisition of SNV between sexes, group foraging, and various other factors may expand our understanding of the risk factors for acquiring HPS. Taken together and applied, we anticipate developing methods for preventing this disease as well as diseases caused by other rodent-borne viruses.

Adolescent↗
PubMed1999

A genetically engineered spleen necrosis virus-derived retroviral vector that displays the HIV type 1 glycoprotein 120 envelope peptide.

We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.

Animals↗
PubMed2003

Prognosis and outcome of 26 patients with systemic necrotizing vasculitis admitted to the intensive care unit.

OBJECTIVES: To investigate presenting features, prognostic factors and outcomes of patients with systemic necrotizing vasculitis (SNV) admitted to the intensive care unit (ICU). METHODS: We retrospectively reviewed the medical records of all 210 SNV patients followed in our university hospital and admitted to the ICU between 1982 and 2001, with respect to clinical features, ICU disease severity scores (APACHE II and SAPS II), the Birmingham vasculitis activity score (BVAS), the five-factors score (FFS) and outcomes. RESULTS: Twenty-six patients (16 men, 10 women) with a mean age of 46.3+/-16.5 yr were included. The reasons for ICU admission were: active SNV, 20 (77%); infection, 3 (12%); others, 3 (12%). SNV was diagnosed in 11 (42%) patients in the ICU. The mean APACHE II and SAPS II scores were significantly higher for patients who died in the ICU (P = 0.01 and P = 0.01 respectively). After a mean follow-up of 31.4+/-29.2 months, the overall mortality rate was 39% (10 patients). Among patients admitted to the ICU with active SNV, BVAS calculated at ICU admission was significantly higher for non-survivors at the end of follow-up (26.9+/-13.0 vs 14.7+/-4.6, P = 0.02). CONCLUSION: The main reason for admitting SNV patients to the ICU was active vasculitis, which was often the first manifestation of SNV and led to its diagnosis. ICU disease severity scores at admission were associated with mortality in the ICU but did not predict long-term outcome, unlike BVAS, which accurately predicted long-term outcome but not ICU prognosis for patients admitted to the ICU with active SNV.

Adolescent↗
PubMed2005

Peptide antagonists that inhibit Sin Nombre virus and hantaan virus entry through the beta3-integrin receptor.

Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the alpha(v)beta(3) integrin, and transfection of human beta(3) integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-beta(3) antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified alpha(v)beta(3), we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 mug/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use beta(3) integrins and PHV uses a different receptor, beta1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via beta(3) integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

Amino Acid Sequence↗
PubMed2005

Cooperative effect of gag proteins p12 and capsid during early events of murine leukemia virus replication.

The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

Capsid Proteins↗
PubMed1994

[Does indocyanine green angiography aid in deciding indications for laser therapy of exudative senile macular degeneration?].

BACKGROUND: Only a few patients with neovascularized age-related macular degeneration (AMD) have clinical and fluorescein angiographic manifestations that meet eligibility criteria for photocoagulation therapy. Angiography with indocyanine green (ICG) may expand those criteria. MATERIAL AND METHODS: Fifty-two patients with exudative AMD were studied by fluorescein and ICG angiography (using the Rodenstock scanning laser ophthalmoscope). The ICG-angiograms were studied in relation to the questions, how often subretinal neovascularizations (SNV) were visible and how often the results were helpful for laser treatment. RESULTS: In 18 cases (about one third) a SNV was detected by ICG-angiography and in 8 (15%) the result allowed a laser treatment. "Ideal" SNV presented as neovascular trees with a visible feeder-vessel. Other cases showed a circumscribed area of diffuse hyperfluorescence especially in the late phase. Macular edema often prevented the resolution of subretinal membranes. CONCLUSIONS: Only randomised studies will be able to show whether the combination of fluorescein and ICG angiography allows the possibility of treating more patients with SNV than fluorescein angiography alone. In the following circumstances this might be the case: 1. If in subfoveal SNV a feeder-vessel could be delineated with ICG-angiography that might be treated selectively. 2. If an occult SNV might be reclassified as manifest SNV due to findings provided by ICG-angiography. 3. Examination of patients with ICG-angiography immediately after laser photocoagulation might be a way of proving the completeness of the treatment.

Aged↗
PubMed1999

Sin Nombre virus pathogenesis in Peromyscus maniculatus.

Sin Nombre virus (SNV), a member of the Hantavirus genus, causes acute viral pneumonia in humans and is thought to persistently infect mice. The deer mouse, Peromyscus maniculatus, has been identified as the primary reservoir host for SNV. To understand SNV infection of P. maniculatus, we examined wild deer mice for localization of viral antigens and nucleic acid. Morphologic examination consistently revealed septal edema within lung tissue and mononuclear cell infiltrates in portal areas of the liver. Immunohistochemical analysis of SNV-infected deer mice identified viral antigens within lung, liver, kidney, and spleen. The lungs consistently presented with the highest levels of viral antigen by immunohistochemistry and with the highest levels of nucleic acid by reverse transcriptase (RT) PCR. The mononuclear cell infiltrates surrounding liver portal triads were positive for SNV antigens in addition to resident macrophages in liver sinuses. Spleen tissue contained antigens in both the red pulp and the periartereolar region of the white pulp. The kidney presented with no gross pathology, although antigens could be localized to glomeruli. Virus antigen levels within the kidney were highest in deer mice that did not have antibodies to SNV but contained viral nucleic acid detectable by RT PCR. Since transmission is thought to occur via urine, our results suggest that virus transmission may be highest in the early stages of infection. In addition, these results indicate that SNV does cause some pathology within its reservoir host.

Animals↗
PubMed2002

cis-Acting elements important for retroviral RNA packaging specificity.

Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Psi and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Psi. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5'-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

3T3 Cells↗
PubMed2002

Nuclear interactions are necessary for translational enhancement by spleen necrosis virus RU5.

The 5' long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5' RNA terminus, functions in a position- and orientation-dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.

5' Untranslated Regions↗