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PubMed1997

Molecular determinants for virulence in coxsackievirus B1 infection.

Studies demonstrated that a strain derived from an infectious clone of coxsackievirus B1 (CVB1N) (N. Iizuka, H. Yonekawa, and A. Nomoto, J. Virol. 65:4867-4873, 1991) was 3 to 4 log10 less virulent than the myotropic Tucson strain of CVB1 (CVB1T) following intraperitoneal inoculation of newborn mice. Replacement of nucleotides (nt) 69 to 804 from the 5' untranslated region (5' UTR) and 1A coding region of CVB1N or nt 117 to 161 from the 5' UTR with the corresponding part from CVB1T restored greater than 90% of the virulence. Sequencing of the 5' UTR of CVB1T demonstrated areas with a greater similarity to particular echoviruses than to CVB1N, suggesting that recombination events might have occurred, perhaps influencing the virulence phenotype.

Animals↗

PubMed1998

Distinct mechanisms trigger apoptosis in human immunodeficiency virus type 1-infected and in uninfected bystander T lymphocytes.

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4+ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4+ or CD8+ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4+ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4+ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.

Antigen-Presenting Cells↗

PubMed1998

Mutational and functional analysis of the C-terminal region of the C3H mouse mammary tumor virus superantigen.

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vbeta) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vbeta chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vbeta14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.

Amino Acid Sequence↗

PubMed1998

Sequences in pol are required for transfer of human foamy virus-based vectors.

A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis-acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5' end upstream of position 1274 of the proviral DNA. Interestingly, a mutation in the leader sequence which decreased the ability to dimerize in vitro inhibited transfer by helper HFV. A second element that was important for vector transfer was located in the pol gene between positions 5638 and 6317. Constructs lacking this element were only poorly transferred by helper HFV, even though their RNA was produced in the vector cell lines. This finding rules out the possibility that the observed lack of transfer was due to RNA instability. A minimal vector containing only these two elements could be successfully delivered by helper HFV, confirming that all essential cis-acting sequences were present. The presence of a sequence described as a second polypurine tract in HFV was not necessary for transfer. Our data identified the minimal sequence requirements for HFV vector transfer for the development of useful vector systems.

Animals↗

PubMed1999

Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1.

Barley yellow dwarf luteovirus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.

Base Sequence↗

PubMed1999

Mutagenic analysis of the 3' cis-acting elements of the rubella virus genome.

Thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) structures; SL1 and SL2 are both located in the E1 coding region, while SL3 and SL4 are within the 59-nt 3' untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis. RNase mapping indicated that SL2 and SL3 are in equilibrium between two conformations, in the second of which the previously proposed CAL binding site in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3' UTR with a RUB infectious clone, Robo302, revealed that most of the 3' UTR is required for viral viability except for the 3'-terminal 5 nt and the poly(A) tract, although poly(A) was rapidly regenerated during subsequent replication. Maintenance of the overall SL3 structure, the 11-nt single-stranded sequence between SL3 and SL4, and the sequences forming SL4 were all important for viral viability. Studies on the interaction between host factors and the 3' UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced cross-linking detected six host protein species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, interacting with the 3' UTR. Site-directed mutagenesis of SL2 by nucleotide substitutions showed that maintenance of SL2 stem rather than the U-U bulge was critical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants having the SL2 stem destabilized had much lower binding activity. However, all of these mutations gave rise to viable viruses when introduced into Robo302, indicating that binding of CAL to SL2 is independent of viral viability.

Base Sequence↗

PubMed1999

Gene therapy vectors based on adeno-associated virus type 1.

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.

Adolescent↗

PubMed1999

The H gene of rodent brain-adapted measles virus confers neurovirulence to the Edmonston vaccine strain.

Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.

Adaptation, Biological↗

PubMed1999

Simian immunodeficiency virus (SIV) from sun-tailed monkeys (Cercopithecus solatus): evidence for host-dependent evolution of SIV within the C. lhoesti superspecies.

Recently we reported the characterization of simian immunodeficiency virus (SIVlhoest) from a central African l'hoest monkey (Cercopithecus lhoesti lhoesti) that revealed a distant relationship to SIV isolated from a mandrill (SIVmnd). The present report describes a novel SIV (SIVsun) isolated from a healthy, wild-caught sun-tailed monkey (Cercopithecus lhoesti solatus), another member of the l'hoest superspecies. SIVsun replicated in a variety of human T-cell lines and in peripheral blood mononuclear cells of macaques (Macaca spp.) and patas monkeys (Erythrocebus patas). A full-length infectious clone of SIVsun was derived, and genetic analysis revealed that SIVsun was most closely related to SIVlhoest, with an amino acid identity of 71% in Gag, 73% in Pol, and 67% in Env. This degree of similarity is reminiscent of that observed between SIVagm isolates from vervet, grivet, and tantalus species of African green monkeys. The close relationship between SIVsun and SIVlhoest, despite their geographically distinct habitats, is consistent with evolution from a common ancestor, providing further evidence for the ancient nature of the primate lentivirus family. In addition, this observation leads us to suggest that the SIVmnd lineage should be designated the SIVlhoest lineage.

Amino Acid Sequence↗

PubMed2000

Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates.

A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log(10) PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log(10) focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log(10) FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.

Amino Acid Substitution↗

PubMed2000

Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice.

Several alphaviruses, including the Sindbis-group viruses, Ross River virus, O'nyong-nyong virus, and Chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. Mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. S.A.AR86 is a Sindbis-group alphavirus closely related to the arthralgia-associated Ockelbo and Girdwood S.A viruses. Following inoculation with S.A.AR86 derived from a molecular clone, infectious virus was isolated from bone and joint tissue 1 to 6 days postinfection. Studies using either in situ hybridization or S.A.AR86-derived double promoter viruses and replicons expressing green fluorescent protein localized sites of viral replication to the periosteum, tendons, and endosteum within the epiphyses of the long bones adjacent to articular joints. These results demonstrate that alphaviruses associated with arthralgia in humans replicate within bone-associated connective tissue adjacent to articular joints in an adult mouse model.

Alphavirus↗

PubMed2000

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

Animals↗

PubMed2000

A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86.

S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c. ) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.

Alphavirus↗

PubMed2001

A predicted secondary structural domain within the internal ribosome entry site of echovirus 12 mediates a cell-type-specific block to viral replication.

The enterovirus 5' nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5' (m(7)GpppN) cap-independent manner, and cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5' NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5'NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37 degrees C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5'NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5' NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.

5' Untranslated Regions↗

PubMed2001

Latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus interacts with Tat and activates the long terminal repeat of human immunodeficiency virus type 1 in human cells.

The latency-associated nuclear antigen (LANA) is constitutively expressed in cells infected with the Kaposi's sarcoma (KS) herpesvirus (KSHV), also referred to as human herpesvirus 8. KSHV is tightly associated with body cavity-based lymphomas (BCBLs) in immunocompromised patients infected with human immunodeficiency virus (HIV). LANA, encoded by open reading frame 73 of KSHV, is one of a small subset of proteins expressed during latent infection and was shown to be important in tethering the viral episome to host chromosomes. Additionally, it has been shown that LANA can function as a regulator of transcription. However, its role in the progression of disease is still being elucidated. Since KS is one of the most common AIDS-associated cancers in the United States and BCBLs appear predominantly in AIDS patients, we examined whether LANA is able to regulate the HIV type 1 (HIV-1) long terminal repeat (LTR). Using luciferase-based transient transfection assays, we found that LANA was able to transactivate the HIV-1 LTR in the human B-cell line BJAB, human monocytic cell line U937, and the human embryonic kidney fibroblast cell line 293T. Moreover, we observed that the virus-encoded HIV transactivator protein Tat cooperated with LANA in activation of the LTR in a dose-response fashion with increasing amounts of LANA. Surprisingly, LANA alone was sufficient to transactivate the HIV-1 LTR in BJAB cells. In similar assays using a HIV-1 LTR construct with the core enhancer elements deleted; the activity of LANA was diminished but not abolished, indicating a mechanism which involves the cooperation of the core enhancer elements and downstream elements which include Tat. Furthermore, transient transfection of an infectious clone of HIV with LANA demonstrated effects similar to those seen in the reporter assays based on Western blot analysis of HIV Gag polypeptide p24. Interestingly, we also demonstrated that the carboxy terminus of LANA associates with Tat in cells and in vitro. These experiments suggest a role for LANA in activating the HIV-1 LTR through association with cellular molecules targeting the core enhancer elements and Tat and may have important consequences in increasing the levels of HIV in infected individuals and, hence, the disease state.

Antigens, Viral↗

PubMed2001

Hepatitis C virus 3'X region interacts with human ribosomal proteins.

To identify proteins that can bind the 3' untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3'-terminal 98 nucleotides (3'X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3'X region and these proteins by a three-hybrid mating assay using mutant 3'X sequences. We have further characterized the interaction between 3'X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3'X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3'X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase [GST] fusion protein) together with a 3'X riboprobe. The 3'X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3'X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.

3' Untranslated Regions↗

PubMed2002

Characterization of the transcription profile of adeno-associated virus type 5 reveals a number of unique features compared to previously characterized adeno-associated viruses.

We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7- and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.

Dependovirus↗

PubMed2003

Reverse genetic system for the analysis of parvovirus telomeres reveals interactions between transcription factor binding sites in the hairpin stem.

The left-hand or 3'-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.

Animals↗