Juan Carlos de la Torre

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance-a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

Persistent LCMV Infection Is Controlled by Blockade of Type I Interferon Signaling

INTERFER(ON)ing Persistence During persistent viral infections, a dysregulated immune response fails to control the infection. Wilson et al. (p. 202) and Teijaro et al. (p. 207; see the Perspective by Odorizzi and Wherry) show this occurs because type I interferons (IFN I), critical for early responses to viral infection, contribute to the altered immunity seen during persistent infection. Antibody blockade of IFN I signaling during chronic lymphocytic choriomeningitis virus (LCMV) in mice resulted in reduced viral titers at later stages of infection, reduced expression of inhibitory immune molecules and prevented the disruptions to secondary lymphoid organs typically observed during persistent infection with LCMV. Whether type I IFNs are also detrimental to persistent viral infection humans, such as HIV and hepatitis C virus, remains to be determined. Blockade of type I interferons leads to better control of persistent lymphocytic choriomeningitis virus infection. [Also see Perspective by Odorizzi and Wherry] During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell–dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.

The small RING finger protein Z drives arenavirus budding: Implications for antiviral strategies

By using a reverse genetics system that is based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we have identified the arenavirus small RING finger Z protein as the main driving force of virus budding. Both LCMV and Lassa fever virus (LFV) Z proteins exhibited self-budding activity, and both substituted efficiently for the late domain that is present in the Gag protein of Rous sarcoma virus. LCMV and LFV Z proteins contain proline-rich motifs that are characteristic of late domains. Mutations in the PPPY motif of LCMV Z severely impaired the formation of virus-like particles. LFV Z contains two different proline-rich motifs, PPPY and PTAP, which are separated by eight amino acids. Mutational analysis revealed that both motifs are required for efficient LFV Z-mediated budding. Both LCMV and LFV Z proteins recruited to the plasma membrane Tsg101, which is a component of the class E vacuolar protein sorting machinery that has been implicated in budding of HIV and Ebola virus. Targeting of Tsg101 by RNA interference caused a strong reduction in Z-mediated budding. These results indicate that Z is the arenavirus functional counterpart of the matrix proteins found in other negative strand enveloped RNA viruses. Moreover, members of the vacuolar protein sorting pathway appear to play an important role in arena-virus budding. These findings open possibilities for antiviral strategies to combat LFV and other hemorrhagic fever arenaviruses.

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

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.

Inhibition of the Type I Interferon Response by the Nucleoprotein of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

ABSTRACT The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-β) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.

NP and L Proteins of Lymphocytic Choriomeningitis Virus (LCMV) Are Sufficient for Efficient Transcription and Replication of LCMV Genomic RNA Analogs

ABSTRACT The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation ofcis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5′ untranslated region (UTR) and IGR and downstream by the S 3′ UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viraltrans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.

Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV).

Passage of the prototypic arenavirus lymphocytic choriomenigitis virus (LCMV) in cultured cells in the presence of the mutagenic agent 5-fluorouracil (FU) resulted in efficient and systematic virus extinction under conditions that did not significantly affect cell survival. FU-mediated extinction of LCMV was associated with 3.6- to 10-fold increases in the mutation frequencies for the three viral genes examined, but with only very modest effects on virus replication and transcription during a single round of infection. Likewise, FU did not affect expression of a LCMV minigenome. In contrast, the well documented antiviral effect of ribavirin against LCMV was not associated with significant increases in virus mutation frequencies, but rather with a dramatic inhibition of both viral RNA synthesis and LCMV minigenome expression. Mutagen induced viral extinction has been recently reported for positive strand RNA viruses polio and foot-and-mouth disease, and the lentivirus HIV-1. Our findings indicate that lethal mutagenesis can be effective also against LCMV, a negative strand RNA virus. Moreover, FU treatment prevented the establishment of LCMV persistent infection in mice deficient in B and T cells, suggesting the feasibility in vivo of lethal mutagenesis as a novel antiviral strategy.

Differential inhibition of type I interferon induction by arenavirus nucleoproteins.

ABSTRACT We have documented that the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus is an antagonist of the type I interferon response. In this study we tested the ability of NPs encoded by representative arenavirus species from both Old World and New World antigenic groups to inhibit production of interferon. We found that, with the exception of Tacaribe virus (TCRV), all NPs tested inhibited activation of beta interferon and interferon regulatory factor 3 (IRF-3)-dependent promoters, as well as the nuclear translocation of IRF-3. Consistent with this observation, TCRV-infected cells also failed to inhibit interferon production.

Ube1L and protein ISGylation are not essential for alpha/beta interferon signaling.

ABSTRACT The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L−/− mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L−/− mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-α/β signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.

RING Finger Z Protein of Lymphocytic Choriomeningitis Virus (LCMV) Inhibits Transcription and RNA Replication of an LCMV S-Segment Minigenome

ABSTRACT Arenaviruses have a bisegmented negative-strand RNA genome whose proteomic capability is limited to only four polypeptides, namely, nucleoprotein (NP), surface glycoprotein (GP) that is proteolytically processed into GP1+GP2, polymerase (L), and a small (11-kDa) RING finger protein (Z). The role of Z during the Lymphocytic choriomeningitis virus (LCMV) life cycle is poorly understood. We investigated the function of Z in virus transcription and replication by using a reverse genetic system for the prototypic arenavirus LCMV. This system involves an LCMV minigenome and the minimal viral trans-acting factors (NP and L), expressed from separated cotransfected plasmids. Cotransfection of the Z cDNA strongly inhibited LCMV minigenome expression. The effect required synthesis of Z protein; its magnitude was dose dependent and occurred with levels of Z protein substantially lower than those observed in LCMV-infected cells. Coexpression of Z did not prevent the encapsidation of plasmid supplied minigenome, but it affected both transcription and RNA replication similarly. Mutations in Z that unfolded its RING finger domain eliminated its inhibitory activity, but RING proteins not related to Z did not affect LCMV minigenome expression. Consistent with the minigenome results, cells transiently expressing Z exhibited decreased susceptibility to infection with LCMV.