Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing 1a and 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5',3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable of viral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

Infection of cells by varicella zoster virus: inhibition of viral entry by mannose 6-phosphate and heparin.

Envelope glycoproteins of varicella zoster virus (VZV) contain mannose 6-phosphate (Man6P) residues. We now report that Man6P competitively and selectively inhibits infection of cells in vitro by cell-free VZV; furthermore, dephosphorylation of VZV by exposure to alkaline phosphatase rapidly destroys infectivity. Cells are also protected from VZV in a concentration-dependent manner by heparin (ED50 = 0.23 micrograms/ml; 95% confidence limits = 0.16-0.26 microgram/ml) but not by chondroitin sulfate. Both heparin and Man6P are protective only when present about the time of inoculation. Heparin but not Man6P interferes with the attachment of VZV to cell surfaces; moreover, VZV binds to heparin-affinity columns. These data are compatible with a working hypothesis, whereby VZV attaches to cell surfaces by binding to a heparin sulfate proteoglycan. This binding stabilizes VZV, making possible a low-affinity interaction with another Man6P-dependent receptor, which is necessary for viral entry.