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Background: Hepatitis C infection (HCV) and hepatocellular carcinoma (HCC), the two main causes of liver transplantation (LT), have reduced survival post-LT. The impact of HCV, HCC and their coexistence on post-LT survival were assessed. Methodology: All 601 LT patients from 1992 to 2011 were reviewed. Those deceased within 30 days (n = 69) and re-transplants (n = 49) were excluded. Recipients were divided into four groups: (a) HCC-/HCV-(n = 252) (b) HCC+/HCV-(n = 58), (c) HCC-/HCV+ (n = 106) and (d) HCC+/HCV+ (n = 67). Demographics, the donor risk index (DRI), Model for End-Stage Liver Disease (MELD) score, survival, complications and tumour characteristics were collected. Statistical analysis included anova, chi-square, Fisher's exact tests and Cox and Kaplan–Meier for overall survival. Results: Groups were comparable with regards to baseline characteristics, but HCC patients were older. After adjusting for age, MELD, gender and the donor risk index (DRI), survival was lower in the HCC+/HCV+ group (59.5% at 5 yrs) and the hazard ratio (HR) was 1.90 [95% confidence interval (CI),1.24–2.95, P = 0.003] and 1.45 (95% CI, 0.99–2.12, P = 0.054) for HCC-/HCV+. HCC survival was similar to controls (HR 1.18, 95% CI, 0.71–1.93, P = 0.508). HCC+/HCV-patients exceeded the Milan criteria (50% versus 31%, P < 0.04) and had more micro-vascular invasion (37.5% versus 20.6%, P = 0.042). HCC+/HCV+ versus HCC+/HCV-survival remained lower (HR 1.94, 95% CI, 1.06–3.81, P = 0.041) after correcting for tumour characteristics and treatment. Conclusion: HCV patients had lower survival post-LT. HCC alone had no impact on survival. Patient survival decreased in the HCC+/HCV+ group and this appears to be as a consequence of HCV recurrence.

We propose that a nucleotide template-based mechanism facilitates the acquisition of the K65R mutation in subtype C human immunodeficiency virus type 1 (HIV-1). Different patterns of DNA synthesis were observed using DNA templates from viruses of subtype B or C origin. When subtype C reverse transcriptase (RT) was employed to synthesize DNA from subtype C DNA templates, preferential pausing was seen at the nucleotide position responsible for the AAG-to-AGG K65R mutation. This did not occur when the subtype B RT and template were used. Template factors can therefore increase the probability of K65R development in subtype C HIV-1.

Conferences on Antiviral Research present a wealth of new information on novel drug development and the 21st annual meeting, held in Montreal between 13–17 April 2008, was no exception. The sessions topics included novel targets for HIV therapy, respiratory and emerging viruses, hepatitis viruses, clinical update on antiviral drugs, retroviruses, and herpes- and poxviruses, among others. In addition, there were a number of themed poster sessions dealing with each of the above areas, as well as novel methods for antiviral analysis, topical microbicides, medicinal chemistry, animal models, veterinary viruses and assorted viral agents. Each of these sessions were well organized and provoked thoughtful discussions. The session on novel targets for HIV therapy featured cutting-edge lectures on interactions between viral proteins and cellular factors. This work has the potential to lead to novel drug discovery as does that on peptide-based approaches to prevent replication of influenza viruses and the development of novel compounds that interfere with the neuraminidase enzymes of both influenza and parainfluenza viruses. Other areas of focus included novel approaches aimed at heightening immune effector mechanisms and the development of gene therapy strategies for certain types of viral infection. Of course, the development of compounds directed at numerous hepatitis C targets also featured prominently at the meeting.

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

We wished to investigate the effects of various mutations in HIV-1 reverse transcriptase (RT) on biochemical inhibition by the active form of a novel nucleoside termed apricitabine. Accordingly, we studied the efficiency of chain-termination mediated by apricitabine triphosphate (TP) in cell-free assays that used either recombinant wild-type or mutated RTs. We also performed steady-state-kinetics and primer-unblocking assays. Subtype C RTs were also analysed. The results showed that the K65R mutation in RT caused reductions in the efficiency of chain-termination of apricitabine-TP by increasing its Ki. However, K65R did not affect rates of primer unblocking for apricitabine-TP. No significant differences were found between subtype C and subtype B RTs with regard to any of the parameters studied. Other mutations such as M184V, L74V and K103N had no effect on the efficiency of chain termination by apricitabine-TP. Thus, the mechanism of reduced susceptibility to apricitabine of viruses containing K65R in RT seems to be mediated exclusively through a reduction in binding or incorporation of apricitabine-TP. Unlike some other nucleoside analogues, increased excision of incorporated apricitabine does not seem to be a cause of resistance to apricitabine.

Objectives: We recently reported the preferential selection of the K65R resistance mutation in subtype C HIV-1 compared with subtype B and showed the underlying mechanism to be dependent on subtype C-specific silent nucleotide polymorphisms, i.e. genomic mutations that change the genotype but not the phenotype. The number of clinical reports demonstrating elevated numbers of K65R nevertheless suggests the existence of factors limiting the increased incidence of K65R mutations. Thus, we investigated the contributions of subtype C-specific silent nucleotide polymorphisms at thymidine analogue mutation (TAM) sites 70, 210 and/or 219 that might reduce the previously described preferential selection of K65R in subtype C HIV-1 associated with subtype C-specific nucleotide polymorphisms at sites 64/65. Methods: Cell culture drug selections were performed with various drugs in MT2 cells. Results: The use of nucleoside/nucleotide reverse transcriptase inhibitors [N(t)RTIs] as single drugs or in combination confirmed the more frequent selection of K65R by multiple N(t)RTIs in a subtype B virus that contained the 64/ 65 nucleotide polymorphisms of subtype C than in a wild-type subtype B virus. This effect was attenuated in the presence of several silent TAM nucleotide polymorphisms, except when stavudine was employed in the selection protocol. Conclusions: These results further demonstrate that stavudine can preferentially select for K65R in subtype C virus and also provide a basis for understanding the importance of silent nucleotide polymorphisms in regard to altered HIV drug resistance profiles.

Background: The host protein APOBEC3G (A3G) can limit HIV-1 replication. Its protective effect is overcome by the HIV-1 ‘viral infectivity factor' (Vif), which targets A3G for proteosomal degradation. Although Vif is considered to be essential for HIV-1 replication, the effect of Vif variability among commonly used HIV-1 molecular clones of different genetic backgrounds on viral infectiousness and pathogenesis has not been fully determined. Methods: We cloned the intact Vif coding regions of available molecular clones of different subtypes into expression vectors. Δvif full-length HIV-1 clonal variants were generated from corresponding subtype-specific full-length molecular clones. Replication-competent viruses were produced in 293T cells in the presence or absence of A3G, with Vif being supplied by the full-length HIV-1 clone or in trans. The extent of A3G-mediated restriction was then determined in a viral replication assay using a reporter cell line. Results and Conclusions: In the absence of A3G, Vif subtype origin did not impact viral replication. In the presence of A3G the subtype origin of Vif had a differential effect on viral replication. Vif derived from a subtype C molecular clone was less effective at overcoming A3G-mediated inhibition than Vif derived from either subtype B or CRF02_AG molecular clones.

Background:We have shown that the K65R resistance mutation in HIV type-1 (HIV-1) reverse transcriptase (RT) is selected more rapidly in subtype C than subtype B HIV-1 in biochemical, cell culture and clinical studies. Template-usage experiments demonstrated that subtype C nucleotide coding sequences caused RT to preferentially pause, leading to K65R acquisition. This new study now further establishes the basis for differential occurrence of both K65R and thymidine analogue mutations (TAMs) between subtypes.Methods:Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65.Results:When positive double-stranded DNA synthesis was evaluated from a negative single-stranded DNA template, pausing at the 67 region, which is linked to occurrence of TAMs, was alleviated with both subtype B and C templates at high dCTP concentrations, but this alleviation was more pronounced with the subtype C template. By contrast, pausing at the 65 region on the subtype C but not subtype B template always occurred and was not alleviated at high levels of nucleotide triphosphates or by other means. Furthermore, templates containing repeats of the homopolymeric sequence spanning codons 64–66 of pol showed corresponding pausing repeats at the 65 region with the subtype C template only. Inverted RNA and DNA templates both displayed pausing at position K65 for the subtype C template and a ladder of pausing events culminating at codon 67 for the subtype B templates.Conclusions:These results further establish a mechanistic basis for the exclusion of both K65R and TAMs on single templates as well as the preferential acquisition of K65R in subtype C viruses.

Recently, we described a novel nucleotide template-based mechanism that may be the basis for the facilitated acquisition of the K65R resistance mutation in subtype C versus subtype B human immunodeficiency virus type 1 (HIV-1). In this article, we evaluated the effects of subtype C-specific silent polymorphisms in cell culture drug-selection experiments using nucleoside and nucleotide reverse-transcriptase inhibitors. The K65R pathway was selected more frequently in a subtype B virus that contained subtype C nucleotide polymorphisms at both positions 64 and 65 than in a wild-type NL4-3 subtype B virus. This is the first demonstration of the significance of silent nucleotide polymorphisms in the development of drug resistance.

Background: We have shown that the K65R mutation is selected more rapidly in subtype C than in subtype B HIV-1 isolates in both cell culture and clinical studies. Biochemical comparisons between subtype B and C-derived reverse transcriptase (RT) enzymes revealed similar molecular characteristics that do not explain the more rapid selection of K65R with subtype C viruses. This study attempts to establish the mechanistic basis for the difference. Methods: Recombinant subtype C and B HIV-1 RT enzymes were expressed and purified in E. coli. Gel-based nucleotide extension assays were used to study DNA synthesis from various natural and synthetic DNA and RNA templates that spanned regions of the pol gene responsible for the K65R and M184V mutations. Cell based experiments were performed using MT2 cells infected with mutated subtype B HIV-1 pNL4-3 viruses. Results: The propensity for the more rapid selection of K65R with subtype C enzymes is due to the mechanism of DNA synthesis from a subtype C template. The use of templates containing the 64, 65 and 66 codons of the pol gene led to different patterns of DNA synthesis. When subtype C RT was employed to synthesize DNA from subtype C templates, preferential pausing was seen at the nucleotide position responsible for the AAG to AGG mutation on codon 65 which gives rise to K65R. In contrast, the use of subtype B RT together with a subtype B template reveals a different pattern of DNA synthesis. When subtype B RT was employed with a subtype C template, DNA synthesis stopped at the exact nucleotide position responsible for K65R. This phenomenon was not observed when subtype C RT was used with a subtype B template. A similar method was employed to investigate if differences exist in the appearance of M184V between subtypes. The results suggest that M184V is not favoured due to its coding sequence and that the propensity for the development of M184V remains the same in subtype B and C HIV. In cell culture, K65R was detected faster in subtype B that has been mutated to include the 64/65 codons of subtype C, when compared to wild-type subtype B HIV. Conclusions: The more rapid emergence of K65R but not M184V in subtype C RT appears to be based on the pol gene coding sequence. These results urge for the analysis of resistance mechanisms to be studied in all HIV subtypes separately and have clinical relevance in regard to the management of subtype C infections.