Alphaviruses are positive-strand RNA viruses that can mediate efficent cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs 1 and 2). Over the past 10 years, the alphavirus RNA replication and packaging machinery has been adapted for expression of heterologous RNAs and proteins in animal cells (for reviews, see refs. 3-6). As transient expression systems, alphaviruses offer several advantages. These include (i) a broad range of susceptible host cells including those of insect, avian, and mammalian origin; (ii) high levels of cytoplasmic RNA and protein expression without splicing; and (iii) the facile construction and manipulation of recombinant RNA molecules using full-length cDNA clones from which infectious RNA transcripts can be generated by in vitro transcription. Two principal strategies are being employed for expression of heterologous sequences: (i) engineering infectious recombinant RNAs that express additional subgenomic RNAs and (ii) replacement of the structural genes to produce self-replicating RNA "replicons" that can be packaged into infectious particles using defective helper RNAs or packaging cell lines. In addition, incorporation of heterologous ligands or receptors into the virion envelope may eventually allow targeting of engineered alphavirus RNAs to specific cell types. This overview briefly discusses the background, methodology, and applications of these alphavirus vector systems, which range from high-level protein production in cell culture to the induction of protective immunity in animals. (Fig; 1). This subgenomic RNA, which can accumulate to levels approaching 106 molecules per cell, is the mRNA for translation of the structural proteins. The synthesis of minus, plus, and subgenomic RNAs is temporally regulated via proteolytic processing of nonstructural polyprotein replicase components by a virus-encoded protease residing in the C-terminal region of nsP2 (8, 9).The structural proteins are initially translated as a polyprotein (NH2-C-E3-E2-6K-E1-COOH) that is processed coand posttranslationally to produce the mature products. Cleavage at the C-E3 site is mediated by a chymotrypsin-like protease activity residing in the C-terminal portion of the C protein. E3 and E2 are initially made as a precursor (called PE2 or P62) that is processed by a furin-like activity late during release of the virus from infected cells. Envelope glycoproteins El and PE2, separated by signal peptidase cleavages, form a heterodimer that migrates through the secretory pathway to the plasma membrane. In the cytoplasm, C-protein subunits complex with the genome RNA to form a nucleocapsid that matures by budding through the plasma membrane, acquiring a lipid bilayer envelope with embedded viral glycoproteins. Infectious Alphavirus cDNA ClonesStudies on the use of alphaviruses as vectors have required the recovery of infectious replication-competent ...

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989–12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.

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Mycobacterium tuberculosis infection of the genitourinary tract is an uncommon disease in renal transplant recipients and presentation is atypical. Genitourinary tuberculosis is associated with graft rejection, and this diagnosis should be considered for renal transplant recipients with unexplained fever and constitutional symptoms.