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Biomedical subjects

R Andino

Publications and source records attributed to R Andino.

29 records · Page 2Linked to original sources
PubMed1997

Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA.

The 5' noncoding region of the poliovirus genome contains RNA structures important for replication and translation. Here we show that two closely related cellular poly(rC) binding proteins (PCBP1 and PCBP2) bind to the terminal cloverleaf structure and facilitate the interaction of the viral protein 3CD (the uncleaved precursor of the protease-polymerase). In addition, these cellular proteins bind to stem-loop IV of the internal ribosomal entry site. The proteins are cytoplasmic and largely associated with ribosomes; they appear to dimerize in solution and to form heterodimers when binding to stem-loop IV. Initiation of viral translation in Xenopus oocytes is strongly inhibited by co-injection of specific antibodies directed against PCBP1 or PCBP2, indicating that the poly(rC) binding proteins may facilitate this process. Furthermore, PCPB-depleted HeLa extracts translate poliovirus RNA inefficiently and the activity is partially restored by addition of recombinant PCBP proteins.

Animals↗
PubMed1996

Replication of poliovirus in Xenopus oocytes requires two human factors.

We described a novel system to study poliovirus replication in Xenopus oocytes. Poliovirus RNA microinjected into Xenopus oocyte initiates a complete cycle of viral replication, yielding a high level of infectious viruses. Two distinct HeLa cell activities are required, one involved in initiation of translation and the other in RNA synthesis. The translation factor is a large cytoplasmic protein or complex, which is specifically used for initiation of poliovirus translation. The replication factor is required at early stages of RNA synthesis. Formation of infectious poliovirus is highly temperature dependent. At temperatures below 27 degrees C, capsid assembly appears to be impaired. The oocyte system described here could be useful in identifying and characterizing viral and cellular factors involved in virus replication.

Animals↗
PubMed1996

Poliovirus recombinants expressing hepatitis B virus antigens elicited a humoral immune response in susceptible mice.

Expression of foreign antigens in the context of poliovirus vectors may provide a plausible approach to vaccine development. Poliovirus recombinants were constructed by fusing preS surface or core HBV proteins to the poliovirus polyprotein as previously described (Andino et al., Science, 265, 1448-1451, 1994). All recombinant viruses replicated with near wild-type efficiency in tissue culture cells and stably expressed high levels of the HBV antigens. The kinetics of recombinant RNA synthesis were indistinguishable from that of wild-type poliovirus. Exogenous proteins were not incorporated into the poliovirus particles, but HBV core proteins self-assembled into 100S particles composed of free HBV core proteins and fusions with poliovirus capsid proteins. Mice susceptible to poliovirus infection were inoculated with recombinant virus and elicited humoral immune responses against the HBV antigens.

Amino Acid Sequence↗
PubMed1994

Engineering poliovirus as a vaccine vector for the expression of diverse antigens.

As a step toward developing poliovirus as a vaccine vector, poliovirus recombinants were constructed by fusing exogenous peptides (up to 400 amino acids) and an artificial cleavage site for viral protease 3Cpro to the amino terminus of the viral polyprotein. Viral replication proceeded normally. An extended polyprotein was produced in infected cells and proteolytically processed into the complete array of viral proteins plus the foreign peptide, which was excluded from mature virions. The recombinants retained exogenous sequences through successive rounds of replication in culture and in vivo. Infection of animals with recombinants elicited a humoral immune response to the foreign peptides.

Amino Acid Sequence↗
PubMed1994

Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses.

To study the basis of cellular latency of human immunodeficiency virus (HIV), we have used a recombinant luciferase-encoding HIV (HXB-Luc) to superinfect nonproductively HIV-1-infected human leukemic cell lines. HXB-Luc contains the Photinus pyralis luciferase gene in place of the nef gene and provides a highly sensitive, simple assay for HIV infection and expression. To circumvent any superinfection block in latently infected cells, we also generated viruses pseudotyped with murine leukemia virus amphotropic envelope (HXB-Luc:ampho). The parental uninfected lines, U937 and A3.01, from which the latently infected cell lines U1 and ACH-2, respectively, were derived could be readily infected with pseudotyped or nonpseudotyped reporter viruses. However, superinfection of U1 cells with either HXB-Luc or HXB-Luc:ampho resulted in only low levels of luciferase activity. Like the endogenous provirus, HXB-Luc provirus could be efficiently activated by phorbol ester treatment of HXB-Luc:ampho-superinfected U1 cells. In contrast, superinfection of ACH-2 cells resulted in active expression of the secondarily introduced virus even in unstimulated cells and luciferase production higher than in the parental cell line A3.01. Thus, the proviral latency in U1 cells appears to result from a defect in the cellular environment (a trans effect), whereas the latency in ACH-2 is specific to the integrated provirus and is probably a cis effect due to the site of integration. These results demonstrate distinct modes of proviral latency in these two cell line models and may have implications in our understanding of the regulation and significance of cellular latency in HIV infection.

Base Sequence↗
PubMed1993

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

Amino Acid Sequence↗
PubMed1991

The long terminal repeat is not a major determinant of the cellular tropism of human immunodeficiency virus type 1.

The long terminal repeats (LTRs) of human immunodeficiency virus type 1 (HIV-1) strains from the central nervous systems of four patients with AIDS and of an HIV-1 isolate which is highly macrophage-tropic were isolated by using the polymerase chain reaction. In transient transfection assays, these LTRs demonstrated no significant difference in basal or stimulated levels of transcription in any of a variety of cell lines tested, compared with expression directed from the LTR of a T-lymphocyte-tropic strain of HIV-1. Chimeric viruses were created with the LTRs of the macrophage-tropic and brain-derived viruses ligated to the viral backbone from a T-lymphocyte-tropic strain. No change in cellular tropism was demonstrated with these chimeric viruses. Thus, unlike the LTRs of some murine retroviruses, the LTR of HIV-1 does not appear to play a major role in determining cellular tropism.

Acquired Immunodeficiency Syndrome↗
PubMed1990

A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA.

The existence of a computer-predicted cloverleaf structure for the first 100 nucleotides at the 5' end of poliovirus RNA was verified by site-directed mutagenesis and by chemical and RNAase probing. Mutations that modified the cloverleaf in the positive strand but not the negative strand were lethal to the virus. This RNA cloverleaf structure binds a cellular protein and the viral proteins 3Cpro and 3Dpol. Mutations in specific regions of the RNA cloverleaf prevented this binding. Mutations in either 3Cpro or the RNA that disrupted ribonucleoprotein complex formation inhibited virus growth and selectively affected positive strand RNA accumulation. Phenotypic reversion of these mutations restored the ability to form the complex. Thus, a cloverleaf structure in poliovirus RNA plays a central role in organizing viral and cellular proteins involved in positive strand production.

Base Composition↗
PubMed1990

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication.

Amino Acid Sequence↗
PubMed1988

An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

Animals↗