Cloning of herpesviral genomes as bacterial artificial chromosomes

The human cytomegalovirus (HCMV) UL82-encoded tegument protein pp71 has recently been shown to activate viral immediate-early (IE) gene expression by neutralizing a cellular intrinsic immune defense instituted by the ND10 protein hDaxx. Pp71 localizes to ND10 upon infection and induces the degradation of hDaxx. Here, we report the successful generation of a recombinant HCMV expressing enhanced yellow fluorescent protein (EYFP) fused to the N terminus of pp71. Intriguingly, insertion of the EYFP-UL82 coding sequence into the HCMV AD169 genome gave rise to a recombinant virus, termed AD169/EYFP-pp71, that replicates to significantly higher titers than wild-type AD169. In particular, we noticed strongly increased protein levels of pp71 after AD169/EYFP-pp71 inoculation. Although the high abundance of pp71 resulted in augmented packaging of the tegument protein into viral particles, no increased hDaxx degradation was detectable upon AD169/EYFP-pp71 infection. In contrast, further investigation revealed a significantly enhanced viral DNA replication compared to wild-type AD169. Thus, we hypothesize that an as-yet-unidentified function of pp71 contributes to the enhanced infectivity of AD169/EYFP-pp71. This assumption is additionally supported by the observation that increased early and late gene expression after AD169/EYFP-pp71 infection occurs independent of elevated IE protein levels. Finally, immunofluorescence analyses confirmed that hDaxx determines the ND10-localization of pp71 upon infection, since pp71 exhibited a nucleolar distribution in the absence of hDaxx. Taken together, we generated a recombinant HCMV that constitutes a useful tool not only to dissect the in vivo dynamics of pp71 subnuclear localization more precisely but also to explore new features of this viral transactivator.

Section: Construction and Verification Of A Recombinant Hcmv Expressimentioning

confidence: 99%

Insertion of an EYFP-pp71 (UL82) Coding Sequence into the Human Cytomegalovirus Genome Results in a Recombinant Virus with Enhanced Viral Growth

Tavalai

Kraiger

Kaiser

et al. 2008

“…This approach was used in our laboratory for the construction of the first CoV infectious cDNA clone (3) and, more recently, for the successful construction of other coronavirus (26) and arterivirus (7) cDNA clones. This system permits the stable maintenance in bacteria of large DNA fragments from a variety of complex genomic sources (1,24). Furthermore, the manipulation of the cDNA clone is similar to that of a conventional plasmid and directly allows the recovery of infectious virus from the cDNA clone without the need for in vitro ligation and transcription steps.…”

mentioning

confidence: 99%

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

Almazán

DeDiego

Galán

et al. 2006

208

284

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.The etiologic agent causing severe acute respiratory syndrome (SARS) is a novel coronavirus (CoV) (8,10,(16)(17)(18)21). This virus causes a life-threatening respiratory disease for which no fully efficacious therapy is available. SARS-CoV is a member of group 2 of the Coronaviridae family within the order Nidovirales (13), which is composed of enveloped, singlestranded, positive-sense RNA viruses relevant in animal and human health (5, 9). Two-thirds of the 29.7-kb SARS-CoV genome carries the replicase gene, which comprises two overlapping open reading frames, ORF 1a and ORF 1b, the latter being translated by a ribosomal frameshift mechanism (29). Translation of both ORFs results in the synthesis of two polyproteins that are processed by viral proteinases to release the components of the replication-transcription complex (36,37). Besides containing RNA-dependent RNA polymerase, RNA helicase, and proteases (4,12,15,23,37), which are all common to positive-strand RNA viruses, the CoV replicase was recently predicted to contain a variety of RNA-processing enzymes that are extremely rare or absent in other RNA viruses, including endoribonuclease (NendoU), 3Ј-to-5Ј exoribonuclease (ExoN), 2Ј-O-ribose methyltransferase (2Ј-O-MT), ADP ribose 1ЈЈ-phosphatase, and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase (25, 36). These enzymatic activities might be involved in the replication of the largest known RNA virus genome and in the production of an extensive set of 5Ј-and 3Ј-coterminal subgenomic RNAs (11,14,25,36).The study of CoV molecular biology has been profoundly advanced by the recent construction of full-length cDNA clones (3,6,26,27,(32)(33)(34) and self-replicating RNAs, or replicons (2,28,30). Due to the large size of the CoV RNA genome and the instability of some CoV replicase gene sequences in bacteria, cDNA clones and replicons have been engineered using bacterial artificial chromosomes (BACs) (3), in vitro ligation of CoV cDNA fragments (32), and vaccinia virus as a vector for the propagation of CoV full-length cDNAs (27). Recently, a SARS-CoV full-length cDNA clone has been generated by the approach of using the in vitro li...

“…The genetic malleability of MHV68 enables the identification of viral genes involved in specific aspects of viral pathogenesis by targeted mutagenesis (1). For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24,26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency maintenance (22,42) or orf72 (v-cyclin) in replication and reactivation (61,68,69).…”

mentioning

confidence: 99%

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

Forrest

Speck

2008

Gammaherpesvirus 68 (␥HV68, or MHV68) is a naturally occurring rodent pathogen that replicates to high titer in cell culture and is amenable to in vivo experimental evaluation of viral and host determinants of gammaherpesvirus disease. However, the inability of MHV68 to transform primary murine B cells in culture, the absence of a robust cell culture latency system, and the paucity of MHV68-positive tumor cell lines have limited an understanding of the molecular mechanisms by which MHV68 modulates the host cell during latency and reactivation. To facilitate a more complete understanding of viral and host determinants that regulate MHV68 latency and reactivation in B cells, we generated a recombinant MHV68 virus that encodes a hygromycin resistance protein fused to enhanced green fluorescent protein as a means to select cells in culture that harbor latent virus. We utilized this virus to infect the A20 murine mature B-cell line and evaluate reactivation competence following treatment with diverse stimuli to reveal viral gene expression, DNA replication, and production of progeny virions. Comparative analyses of parental and infected A20 cells indicated a correlation between infection and alterations in DNA damage signaling following etoposide treatment. The data described in this study highlight the potential utility of this new cell culture-based system to dissect molecular mechanisms that regulate MHV68 latency and reactivation, as well as having the potential of illuminating biochemical alterations that contribute to gammaherpesvirus pathogenesis. In addition, such cell lines may be of value in evaluating targeted therapies to gammaherpesvirus-related tumors.Gammaherpesvirus 68 (␥HV68 or MHV68) is a rodent rhadinovirus found naturally in European bank voles and wood mice (6, 7). As a rodent pathogen, MHV68 infection of laboratory mice offers an experimental system to investigate aspects of gammaherpesvirus pathogenesis. Following experimental inoculation of laboratory mice, MHV68 undergoes productive replication at the site of inoculation, which facilitates dissemination to distal organs and is followed by quiescence or latency, a form of infection where viral gene expression is restricted and progeny virions are not produced (41,51,71). In the case of MHV68, latent infection is established in B lymphocytes, as well as epithelial cells, macrophages, and dendritic cells (19,56,58,74). Latency is maintained for the lifetime of the host, and latently infected cells retain the capacity to reinitiate the productive replication cycle, a process termed reactivation (45, 51, 52).The genetic malleability of MHV68 enables the identification of viral genes involved in specific aspects of viral pathogenesis by targeted mutagenesis (1). For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24, 26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency mai...