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Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.

The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.

Sindbis virus (SIN) is a small positive-strand enveloped RNA The SIN genome consists of a single-stranded mRNA molecule of 11,703 bases with a 5' cap structure and a 3' poly(A) tract. Upon entry of the host cell, the 5' two-thirds of the genomic RNA is translated to produce replicase proteins essential for cytoplasmic RNA synthesis. RNA amplification is initiated by the synthesis of full-length complementary minus-strand RNAs, which serve as templates for synthesis of additional genome-length plus strands. By initiation at an internal promoter sequence, genome-length minus strands are also used for synthesis of 3'-terminal subgenomic mRNAs that encode the SIN structural proteins. In certain vertebrate cell types, the SIN replication machinery can produce an estimated 5 x 105 molecules per cell of the subgenomic mRNA, -W-107-108 molecules per cell of the virion structural proteins, and released virus titers in excess of 103 infectious particles per cell. As a transient expression system for heterologous RNAs and proteins, SIN offers several potential advantages: (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 by using a full-length SIN cDNA clone from which infectious RNA transcripts can be generated by in vitro transcription (2).Previous studies showed that replacement of the SIN structural protein coding region with the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a selfreplicating RNA, transcription of a subgenomic CAT mRNA, and 108 polypeptides per cell of active CAT (3). Since these RNA constructs do not encode structural proteins, their usefulness is limited by the efficiency of RNA transfection or alternatively requires the use of a helper system for packaging (refs. 3 and 4; B. Pragai and C.M.R., unpublished results). Here, we report the construction of SIN vectors that are both replication-packaging competent and that allow the rapid recovery oftMered infectious recombinant virus stocks.