Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primertemplate pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV ؉ 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3 terminus by the 3-to-5 proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3 residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.Poxviruses are large, double-stranded DNA viruses that replicate in the cytoplasm of infected cells. Members of this virus family can cause severe infections, including human smallpox. Smallpox was declared eradicated in 1980, but concerns over bioterrorism (12,27) and a recent outbreak of monkeypox in the midwestern United States (19) illustrate some need for the continued development of effective new treatment regimens. A number of new treatments are currently the subject of active investigation, including immunotherapy. However, antiviral drugs offer a combination of chemical stability and simplicity of delivery that is especially attractive from a public health perspective.One class of drugs that have been shown to inhibit poxvirus replication are the nucleoside phosphonate analogs of cellular deoxyribonucleotides that were developed by De Clercq et al. (9). These drugs have been shown to be effective against a wide range of DNA viruses and retroviruses (reviewed in reference 8), and one of these compounds, (S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl)]cytosine, also known as cidofovir (CDV), has been granted Food and Drug Administration approval for the treatment of cytomegalovirus (CMV)-induced retinitis. CDV has been used off label in the treatment of orf (11) and molluscum contagiosum (21) virus infections. It has also been shown to block ...

The acyclic nucleoside phosphonate drug (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine [(S)-

Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitinproteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitinproteasome system is required during poxvirus infection.

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes, crhB and crhC, which are differentially expressed in the cyanobacterium Anabaena sp. strain PCC 7120. crhC transcription is limited specifically to cold shock conditions while crhB is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5′ untranslated region of crhC revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.

Cidofovir (1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that when CDV is incorporated into a growing primer strand, it can inhibit both the 3′-to-5′ exonuclease and the 5′-to-3′ chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3′-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used 1H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18, and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which was confirmed using NMR monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower Tm for the CDV DNA duplex (46°C) compared to the control (58°C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than the control suggesting increased dynamics around the CDV.