Host ranges of Sf-rhabdoviruses harbored by lepidopteran insects and insect cell lines.

Cell lines derived from Spodoptera frugiperda (Sf), which are the most widely used hosts in the baculovirus-insect cell system, are contaminated with Sf-rhabdoviruses (Sf-RVs). In this study, we identified a closely related virus (Sf-CAT-RV) in the caterpillar species used to isolate the original Sf cell line. We then evaluated the Sf-RV and Sf-CAT-RV host ranges, found Sf-CAT-RV could infect Vero cells, and obtained results suggesting both variants can infect mouse ear fibroblasts. In addition, we found both variants could establish pantropic infections in severely immunocompromised (RAG2/IL2RG-/-) mice. However, both variants were cleared by two weeks post-inoculation and neither produced any symptoms or obvious adverse outcomes in these hosts. We conclude the caterpillars used to isolate Sf21 cells were the most likely source of the Sf-RV contaminant, Sf-RVs and their Sf-CAT-RV progenitor have broader host ranges than expected from previous work, but neither variant poses a serious threat to human health.

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System.

One attractive feature of the baculovirus-insect cell system (BICS) is the baculoviral genome has a large capacity for genetic cargo. This enables construction of viral vectors designed to accept multigene insertions, which has facilitated efforts to produce recombinant multisubunit protein complexes. However, the large genetic capacity of baculovirus vectors has not yet been exploited for multistep pathway engineering. Therefore, we created PolyBac, which is a novel baculovirus shuttle vector, or bacmid, that can be used for this purpose. PolyBac was designed to accept multiple transgene insertions by three different mechanisms at three different sites within the baculovirus genome. After constructing and characterizing PolyBac, we used it to isolate nine derivatives encoding various combinations of up to eight different protein N-glycosylation pathway functions, or glycogenes. We then used these derivatives, which were designed to progressively extend the endogenous insect cell pathway, to assess PolyBac's utility for protein glycosylation pathway engineering. This assessment was enabled by engineering each derivative to produce a recombinant influenza hemagglutinin (rH5), which was used to probe the impact of each glycoengineered PolyBac derivative on the endogenous insect cell pathway. Genetic analyses of these derivatives confirmed PolyBac can accept large DNA insertions. Biochemical analyses of the rH5 products showed each had distinct N-glycosylation profiles. Finally, the major N-glycan on each rH5 product was the predicted end product of the engineered N-glycosylation pathways encoded by each PolyBac derivative. These results generally indicate that PolyBac has utility for multistep metabolic pathway engineering and directly demonstrate that this new bacmid can be used for customized protein glycosylation pathway engineering in the BICS.

Targeting of cucumber necrosis virus coat protein to the chloroplast stroma attenuates host defense response.

Cucumber necrosis virus (CNV) is a (+)ssRNA virus that elicits spreading local and systemic necrosis in Nicotiana benthamiana. We previously showed that the CNV coat protein (CP) arm functions as a chloroplast transit peptide that targets a CP fragment containing the S and P domains to chloroplasts during infection. Here we show that several CP arm mutants that inefficiently target chloroplasts, along with a mutant that lacks the S and P domains, show an early onset of more localized necrosis along with protracted induction of pathogenesis related protein (PR1a). Agroinfiltrated CNV CP is shown to interfere with CNV p33 and Tomato bushy stunt virus p19 induced necrosis. Additionally, we provide evidence that a CP mutant that does not detectably enter the chloroplast stroma induces relatively higher levels of several plant defense-related genes compared to WT CNV. Together, our data suggest that targeting of CNV CP to the chloroplast stroma interferes with chloroplast-mediated plant defense.

A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production.

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell-line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-nodavirus-negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agents. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single-cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five™ cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS.

Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines.

Sf-rhabdovirus was only recently identified as an adventitious agent of Spodoptera frugiperda (Sf) cell lines used as hosts for baculovirus vectors. As such, we still know little about its genetic variation, infectivity, and the potential impact of variation on the Sf-rhabdovirus-host interaction. Here, we characterized Sf-rhabdoviruses from two widely used Sf cell lines to confirm and extend information on Sf-rhabdovirus variation. We then used our novel Sf-rhabdovirus-negative (Sf-RVN) Sf cell line to assess the infectivity of variants with and without a 320bp X/L deletion and found both established productive persistent infections in Sf-RVN cells. We also assessed their infectivity using heterologous insect and mammalian cell lines and found neither established productive persistent infections in these cells. These results are the first to directly demonstrate Sf-rhabdoviruses are infectious for Sf cells, irrespective of the X/L deletion. They also confirm and extend previous results indicating Sf-rhabdoviruses have a narrow host range.

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

Complete Genome Sequence of the Autographa californica Multiple Nucleopolyhedrovirus Strain E2.

Many vectors that are commonly used in the baculovirus/insect cell system (BICS) are derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) strain E2. To facilitate work with these vectors, we sequenced the E2 genome, compared it to that of the AcMNPV C6 strain, and found that they are very similar overall.