Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase α

Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase α

Kaposi’s sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) is linked to the development of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). HHV-6 uses human CD46 as a cellular receptor. Akkapaiboon, X. The Asn-89 site of vIL-6, found to be required for optimal cytokine function, is composed of complex glycans. Furthermore, 17 patients without histopathological GVHD demonstrated a significant lymphoid infiltrate suggesting “pure” HHV-6-related manifestations, and these patients could have been spared steroid therapy. When expressed in mammalian cells, including dendritic cells, γ(1)34.5 associates with IKKα/β and inhibits NF-κB activation. Although herpes primase synthesizes RNA primers 2–13 nucleotides long, the polymerase only effectively elongates those at least 8 nucleotides long.

The observed intrinsic propensity of gB to cluster on membranes indicates an additional role of gB in driving the fusion process forward beyond the transient fusion pore opening and subsequently leading to fusion pore expansion. Fraction of Primers Transferred Intramolecularly—The fraction of primers transferred intramolecularly was determined by measuring the amount of coupled products formed in assays containing binase divided by the amount of coupled products formed in assays lacking binase. Combined, these data demonstrate that gp130, in addition to VKORC1v2, is essential for normal PEL cell growth and survival and that ER-localized vIL-6-gp130 interactions are critical for these activities. shows that binase treatment shortens the full-length products of coupled activity by at least 8 nucleotides, indicating that the herpes polymerase only efficiently elongates primase-synthesized RNA primers >7 nucleotides long to full-length product. 37. Assuming that the full-length products are on average 40 nucleotides long and the average primer length associated with these products is 10 nucleotides long, the rate of full-length product synthesis was 7 nucleotides long (see ). This indicated the epitopes for these MAbs are contained in the external domain of gH, consistent with the MAbs action in neutralization of virion infectivity and inhibition of virus to cell spread by T-lymphocyte fusion.
Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase α

Here, we discover that this escape is mediated by a group of proteins that associates with a sequence element on the IL-6 mRNA. The targeting of HIPK2 into these domains is conditioned by its Ubc9-mediated posttranslational modification with the ubiquitin-like protein SUMO-1 (20). Varying the dNTP concentration from 10 to 90 μm slightly increased the rate of coupled activity and the fraction of primers elongated (around 2-fold in each case, ). KSHV usually exists in a latent state in which a small subset of the viral genome is expressed. Of those primers that the polymerase had elongated, 16% of them were transferred intramolecularly to the dimeric polymerase (UL30-UL42). This is the first report of two kinds of gH-gL complexes on the viral envelope in a member of the herpesvirus family. However, on another DNA template, 3′-d(T20GCCCCAT17)-5′, only 9% of the RNA primers elongated by the polymerase had been passed intramolecularly in assays containing the mutant helicase-primase, whereas 26% were transferred intramolecularly in assays containing the wild-type helicase-primase.

These data suggest that depending upon the template, an active helicase may interfere with direct transfer of primers between the helicase-primase and polymerase. Effect of UL8 on Coupled Activity—To determine whether coupled activity requires UL8, we examined primase-coupled polymerase activity using the minimal helicase-primase complex lacking UL8 (i.e. UL5-UL52). This machinery further includes the receptor binding glycoprotein D (gD) (Montgomery et al., 1996) and the heterodimer of glycoprotein H and L (gH/L) assigned to be the fusion regulator (Chowdary et al., 2010). Thus, to quantitatively compare coupled activity using either UL5-UL52 or UL5-UL52-UL8, assays contained 1 μm [α-32P]dNTPs of a much greater specific activity than if they contained 10 μm dNTPs, although the lower dNTP concentration decreases the efficiency of primer utilization ( and ). Consistent with previous studies (9, 31), adding UL8 to primase assays () greatly stimulated primase activity. Additionally, including an equimolar concentration of UL8 in coupled assays containing UL5-UL52 increased the fraction of primers elongated by UL30-UL42 (∼40–230-fold ()), although this fraction remained pathetically low (99%), pol α utilized 20–30% of the primase-synthesized primers compared with the 1% used by the herpes polymerase ( and ).

Therefore, the claimed solution was obvious. However, comparing primase-coupled polymerase assays containing the UL5-UL52 primase complex versus the UL5-UL52-UL8 complex revealed that the presence of UL8 increased primer utilization by pol α 2–4-fold on two different templates (). These data suggest that UL8 may enhance utilization of UL5-UL52-synthesized primers by pol α. Although in many instances ‘escape’ is likely a reflection of a secondary transcriptional compensation rather than a failure of SOX to cleave the mRNA, a subset of escapees are truly refractory to SOX targeting [12,13].

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