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1.
Figure 2

Figure 2. From: Characterization of human adenovirus 35 and derivation of complex vectors.

Viral DNA synthesis. A549 cells were infected with wt Ad35 at an MOI of 5 focus forming units (FFU) per cell, the viral genome number at each time point was determined by qPCR with primers and probe to pIX coding sequences, standardized to total DNA, and expressed as viral genomes per ng of total DNA. Triplicate infections were performed for each time point; standard deviation error bars shown; hpi = hours post infection.

Duncan McVey, et al. Virol J. 2010;7:276-276.
2.
Figure 4

Figure 4. From: Characterization of human adenovirus 35 and derivation of complex vectors.

Determination of pIX function. (A) Relative abundance of pIX in viral particles. Approximately 1 × 1011 particles of wild type Ad35 or Ad35 with the E1 deletion d2 were fractioned as in Figure 1A and collected for subsequent mass spectroscopy analysis. The analysis of the fraction corresponding to the peak denoted 'IX' in Figure 1A is shown here with the viral sources (Ad35 wt and rAd35E1(d2) indicated. The peak at 14.1 kDa/z is the singly charged protein pIX (1+) and the peak at 7 kDa/z is the doubly charged protein pIX (2+). The abundance of pIX was normalized to hexon abundance because the two proteins co-eluted. Based on intensity the rAd35E1(d2) virus contains ~10% of wild type pIX Ad35 levels. (B) Effect of pIX on heat stability of Ad35 virions. Virions were heat treated in triplicate at 48 C for the time indicated and activity determined by an FFU assay.

Duncan McVey, et al. Virol J. 2010;7:276-276.
3.
Figure 6

Figure 6. From: Characterization of human adenovirus 35 and derivation of complex vectors.

E4 transcriptional analysis. (A) E4 genomic and transcription map with probes used in northern blot analysis (not to scale). Genomic description is as in Figure 5 with the addition of putative splice donor (SD) and splice acceptor (SA) sites. The names of the identified RNAs are given to the right. ^ = splicing; An = polyadenylation.. Base pair coordinates of probes: ORF1 = 34,045-34,412, ORF2 = 33,611-34,015, ORF3 = 33,258-33,611, ORF4 = 32.882-33,214, ORF6 = 32,012-32,887, E4 = 31,855-34,550. (B) Northern blot analysis of RNA from Ad35 wild type-infected 293-ORF6 cells at 8 and 24 hpi. Probes are indicated above each lane and predicted transcripts are labeled on the left of the gel. Numbers on the right denote migration of RNA size standards. (C) Northern analysis at 24 hpi of RNA from wild type Ad35 with the following E4 regions: 1 = wt; 2 = AN; 3 = dORF6. Labeled as in panel B. Identity of transcript h was not determined.

Duncan McVey, et al. Virol J. 2010;7:276-276.
4.
Figure 1

Figure 1. From: Characterization of human adenovirus 35 and derivation of complex vectors.

Identification of Ad35 viral particle proteins. (A) Representative reverse-phase HPLC chromatogram of Ad35 capsid proteins with identities shown. N-term II, C-term II = amino- and carboxy-terminal portions, respectively, of protein II. (B) Ad35 SDS-PAGE protein IV (fiber) identification. Ad35 viruses with wild type (WT) fiber or a genetically modified fiber protein containing the Ad5 fiber knob (5kIV) with predicted molecular masses of 35.4 and 33.9 kDa are indicated by an arrow and arrow head, respectively. Proteins were analyzed by SDS-PAGE and visualized by Deep Purple stain. Molecular weight markers are in the first lane along with their molecular weight in kDa. (C) SDS-PAGE protein assignments for Ad5 and Ad35 capsid proteins stained by silver. Purified virus was loaded at 2.5 × 1010 particles per lane. Molecular weight markers are in the first lane along with their molecular weight in kDa. Ad35 capsid proteins are annotated to the right.

Duncan McVey, et al. Virol J. 2010;7:276-276.
5.
Figure 5

Figure 5. From: Characterization of human adenovirus 35 and derivation of complex vectors.

Effect of E4 deletions on Ad35 growth. (A) Schematic of Ad35 E4 region and deletions (not to scale). The previously identified open reading frames 125R, 145R, 117R, 122R and 299R, are identified by their corresponding Ad5 open reading frame (ORF) ORF1, ORF2, ORF3, ORF4 and ORF6 respectively [6]. The Ad35 ORF6/7 has not been previously described and has coordinates 32,978-32,805 + 32082-31830. The name of the deletion is given to the right with the solid lines indicating which E4 sequences are retained. The description of the junctions are as follows: dORF6 32,010-32,877 with the stop codon of ORF4 changed to TAG, AN 32,007-33,083 with sequence CTAGTCTAGACTAG inserted, dORF3-6 32,010-33,604 with ORF2 stop codon changed to TAG, dORF1-2 33,604-34,416, dORF1-4 32,974-34,416, and ORF3 32,007-33,254 with sequence GCGCGTCGCGA inserted followed by 33,607-34,416. (B and C) Generation of viral progeny on the PC3 cell line. Presence (+) or absence (-) of ORF3 and ORF6 coding sequences is noted below each rAd. Active viral particles were determined at 72 hpi.

Duncan McVey, et al. Virol J. 2010;7:276-276.
6.
Figure 3

Figure 3. From: Characterization of human adenovirus 35 and derivation of complex vectors.

E1B and pIX transcription mapping. (A) Schematic of E1b and pIX sequences, splice junctions shown as ^ with their coordinates that were identified by cDNA analysis, and probe locations (not to scale). Base pair coordinates of the probes are: 1 = 1641-1903, 2 = 2527-3046, 3 = 3283-3359, 4 = 3511-3853. Solid thick lines depict transcripts a, b and c as determined from cDNA and northern blot analysis (panel B) with apparent sizes given to the right in nucleotides (nt). The predicted proteins (boxes) are labeled with the name of their Ad5 homologues pIX, E1b19K, E1b55K [6], and E1b15K [11]. (B) Northern blot analysis of pIX transcripts in cells infected with wild type Ad35. Probes 1, 2, 3, 4 from panel A; a, b, c, = RNAs corresponding to putative mRNAs. (C) Schematic of Ad35 E1 region deletions with Ad35 coordinates corresponding to deletion junctions shown above each line. The viral left ITR, E1A TATAA box, and E1A, E1B, and pIX coding sequences are represented. (D) and (E) Northern blot analysis of transcripts in cells infected with wild type Ad35 (wt) or Ad35 viruses with E1 deletions and hybridized to probe 4. Location of transcripts a, b and c are indicated to the left of each blot.

Duncan McVey, et al. Virol J. 2010;7:276-276.
7.
Figure 7

Figure 7. From: Characterization of human adenovirus 35 and derivation of complex vectors.

Effect of E3 deletions on fiber transcription and virus fitness. (A) Northern blot of 24 hpi total RNA from 293-ORF6 cells infected with wild type Ad35 (wt), E1-deleted rAd35 (d8), or rAd35 d8 constructs with the E3 deletions noted in panel B. The blot was hybridized with a fiber (top) or pIX probe (bottom); the fiber and pIX transcripts are labeled. Numbers on the right denote migration of RNA size standards (kilobases). (B) Schematic of the Ad35 E3 region and E3 deletions (not to scale). The coordinates of the nucleotides that form the deletion junctions are shown above the lines. L4 poly(A), E3 poly(A) = L4 and E3 polyadenylation hexanucleotide signals, respectively. (C) and (D) Viral vector growth competitions. rAd35 d8 E1-deleted vector was mixed with (C) rAd35 d8, E3(X)-deleted vector or with (D) rAd35 d8, E3(HE)-deleted vector. Relative change in genome amounts was determined by DNA restriction fragment analysis of the input mixture of viruses and each serial passage. DNA restriction fragment analysis uniquely identified each virus genome, which is indicated to the left of the gel and their fragment sizes in bp on the right. I = input mixed viruses used for initial infection; M = mock infected cells; P1 = initial infection; P4 = fourth passage; a, b, c = replicates. Restriction enzymes EcoRV and BlpI were used in panels C and D respectively.

Duncan McVey, et al. Virol J. 2010;7:276-276.

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