Salvatore Dimonte

HCV Genotypes Are Differently Prone to the Development of Resistance to Linear and Macrocyclic Protease Inhibitors

Background Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs). Methods The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed. Results Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T). Conclusions The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

Specific HIV-1 integrase polymorphisms change their prevalence in untreated versus antiretroviral-treated HIV-1-infected patients, all naive to integrase inhibitors

OBJECTIVES To define whether the prevalence of mutations associated with integrase inhibitor (INI) resistance is different in untreated versus antiretroviral-treated HIV-1-infected individuals (all INI naive). METHODS Gene sequences of the integrase (IN) and reverse transcriptase (RT) obtained from plasma samples of a well-defined cohort of 448 HIV-1-infected individuals (134 drug naive and 314 antiretroviral treated) were analysed. Docking simulations, using RT and IN models, were also performed. RESULTS Primary mutations and the majority of secondary mutations for raltegravir or elvitegravir were completely absent (or rarely found, <1%) in INI-naive patients, either drug naive or antiretroviral treated. Specific IN polymorphisms increased their frequency in antiretroviral-treated patients, and showed positive associations with specific RT resistance mutations. M154I and V165I IN polymorphisms occurred at a frequency of 6% in untreated patients, reaching 21.3% and 13.4%, respectively, in antiretroviral-treated patients. The mutation M154L, absent in drug-naive patients, was prevalent at 5.7% in antiretroviral-treated patients, and was positively associated with RT resistance mutations F227L and T215Y. Similarly, V165I and G163R mutations were associated with the RT resistance mutations F227L and M230L, respectively, and the T206S polymorphism was associated with the RT resistance mutation L210W. Docking simulations showed several favourable contacts between IN and RT residues. CONCLUSIONS Overall, results confirm that primary and secondary INI-associated mutations are absent or extremely rare in INI-naive patients. Conversely, a few specific IN polymorphisms found in INI-naive patients increased their frequency in antiretroviral-failing patients and/or are associated with RT resistance mutations. The potential contribution of such polymorphisms to the evolution of resistance under the pressure of INIs needs further investigation.

Selected amino acid mutations in HIV-1 B subtype gp41 are Associated with Specific gp120V3 signatures in the regulation of Co-Receptor usage

BackgroundThe third variable loop (V3) of the HIV-1 gp120 surface protein is a major determinant of cellular co-receptor binding. However, HIV-1 can also modulate its tropism through other regions in gp120, such as V1, V2 and C4 regions, as well as in the gp41 protein. Moreover, specific changes in gp41 are likely to be responsible for of damage in gp120-CCR5 interactions, resulting in potential resistance to CCR5 inhibitors.In order to genetically characterize the two envelope viral proteins in terms of co-receptor usage, we have analyzed 526 full-length env sequences derived from HIV-1 subtype-B infected individuals, from our and public (Los Alamos) databases. The co-receptor usage was predicted by the analysis of V3 sequences using Geno2Pheno (G2P) algorithm. The binomial correlation phi coefficient was used to assess covariation among gp120V3 and gp41 mutations; subsequently the average linkage hierarchical agglomerative clustering was performed.ResultsAccording to G2P false positive rate (FPR) values, among 526 env-sequences analyzed, we further characterized 196 sequences: 105 with FPR <5% and 91 with FPR >70%, for X4-using and R5-using viruses, respectively.Beyond the classical signatures at 11/25 V3 positions (S11S and E25D, R5-tropic viruses; S11KR and E25KRQ, X4-tropic viruses), other specific V3 and gp41 mutations were found statistically associated with the co-receptor usage. Almost all of these specific gp41 positions are exposed on the surface of the glycoprotein. By the covariation analysis, we found several statistically significant associations between V3 and gp41 mutations, especially in the context of CXCR4 viruses. The topology of the dendrogram showed the existence of a cluster associated with R5-usage involving E25DV3, S11SV3, T22AV3, S129DQgp41 and A96Ngp41 signatures (bootstrap = 0.88). Conversely, a large cluster was found associated with X4-usage involving T8IV3, S11KRV3, F20IVYV3, G24EKRV3, E25KRV3, Q32KRV3, A30Tgp41, A189Sgp41, N195Kgp41 and L210Pgp41 mutations (bootstrap = 0.84).ConclusionsOur results show that gp120V3 and several specific amino acid changes in gp41 are associated together with CXCR4 and/or CCR5 usage. These findings implement previous observations that determinants of tropism may reside outside the V3-loop, even in the gp41. Further studies will be needed to confirm the degree to which these gp41 mutations contribute directly to co-receptor use.

Specific Enfuvirtide-Associated Mutational Pathways in HIV-1 Gp41 Are Significantly Correlated With an Increase in CD4+ Cell Count, Despite Virological Failure

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) gp41 is a crucial determinant for HIV-1 pathogenicity. We investigated the correlation of enfuvirtide (ENF)-associated gp41 mutational clusters with viroimmunological parameters, as well as the potential underlying mechanisms. METHODS A total of 172 gp41 sequences and clinical follow-up data from 73 ENF-treated patients were analyzed monthly, from baseline to week 48. RESULTS There were 7 novel gp41 mutations positively associated with ENF treatment and correlated with classic ENF mutations. The ENF-associated clusters [V38A + N140I ] and [V 38A +T18A ] significantly correlated with an increase in CD4 cell count at week 48 ( an increase from baseline of 112 and 209 cells/microL, respectively), whereas [Q40H + L45M + 268A] significantly correlated with a decrease in CD4 cell count (-53 cells/microL), without a change in the level of viremia. Residues 38 and 18 are located complementarily to each other in the Rev-responsive element, whereas analysis of molecular dynamics showed that the copresence of [V38A + N140 I] abolishes the interaction between residue 38 and 145 important for stabilization of the 6-helix bundle. In contrast, T268A localizes in the gp41 calmodulin-binding domain responsible for gp41-induced CD4(+) T lymphocyte apoptosis. CONCLUSION Specific gp41 mutational clusters associated with ENF treatment significantly correlate with increases in CD4(+) cell count. Structural analysis suggests that this immunological gain is associated with mechanisms that act at both the protein level and the RNA level (even under conditions of virological failure). This result may help in the selection of patients who can benefit most from ENF treatment and represents a driving force for the design of the next generation of entry inhibitors.

Origin of the 2009 Mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein

Triple-reassortant swine influenza A (H1) viruses, containing genes from avian, human, and swine influenza viruses, emerged and became an outbreak among humans worldwide. Over a 1,000 cases were identified within the first month, chiefly in Mexico and the United States. Here, the phylogenetic analysis of haemagglutin (HA), neuraminidase (NA), and matrix protein (MP) was carried out. The analysis showed that the H1 of this reassortant originated from American pigs, while NA and MP were more likely from European pigs. All of the 2009 isolates appear homogeneous and cluster together, although they are distinct from classical human A (H1N1) viruses.

Identification of the novel KI polyomavirus in the respiratory tract of an Italian patient.

Recently, a new human polyomavirus, KIV, was detected in respiratory specimens of patients with acute respiratory tract infection. Whether this reflects a causal role of the virus in the respiratory tract is still debated. To investigate the presence of KIV in respiratory samples of Italian patients and to determine the degree of similarity with other known polyomaviruses, 222 respiratory specimens collected by general practitioners between 2006 and 2007 were screened. The entire VP1 gene region was amplified and sequenced. Maximum Likelihood tree was generated by PAUP* software. One out of 222 samples tested was positive for KIV. Phylogenetic analysis indicated that this isolate clustered with other KIV isolates, while the WUV isolates seem to belong to a different lineage. The phylogenetic tree also showed that all other known polyomaviruses are quite distant from this isolate. This is the first report describing the presence of KIV in the respiratory tract of a 5‐year‐old Italian child with acute respiratory symptoms. Further investigations are needed to establish an etiological link of KIV with acute respiratory illness. J. Med. Virol. 80:2012–2014, 2008.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Treatment with the fusion inhibitor enfuvirtide influences the appearance of mutations in the human immunodeficiency virus type 1 regulatory protein rev.

ABSTRACT The gp41-encoding sequence of the env gene contains in two separate regions the Rev-responsive elements (RRE) and the alternative open reading frame of the second exon of the regulatory protein Rev. The binding of Rev to the RRE allows the transport of unspliced/singly spliced viral mRNAs out of the nucleus, an essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this study, we have investigated whether the fusion-inhibitor enfuvirtide (ENF) can induce mutations in Rev and if these mutations correlate with the classical ENF resistance gp41 mutations and with viremia and CD4 cell count. Specific Rev mutations were positively associated with ENF treatment and significantly correlated with classical ENF resistance gp41 mutations. In particular, a cluster was observed for the Rev mutations E57A (E57Arev) and N86Srev with the ENF resistance gp41 mutations Q40H (Q40Hgp41) and L45Mgp41. In addition, the presence at week 48 of the E57Arev correlates with a significant viremia increase from baseline to week 48 and with a CD4 cell count loss from baseline to week 48. By modeling the RRE structure, we found that the Q40gp41 and L45gp41 codons form complementary base pairs in a region of the RRE involved in Rev binding. The conformation of this Rev-binding site is disrupted when Q40Hgp41 and L45Mgp41 occur alone while it is restored when both mutations are present. In conclusion, our study shows that ENF pressure may also affect both Rev and RRE structures and can provide an excellent example of compensatory evolution. This highlights the multiple roles of ENF (and perhaps other entry inhibitors) in modulating the correct interplay between the different HIV-1 genes and proteins during the HIV-1 life cycle.

Molecular analysis based on mtLSU-rRNA and DHPS sequences of Pneumocystis jirovecii from immunocompromised and immunocompetent patients in Italy.

Pneumocystis jirovecii is an opportunistic fungus predominantly reported in immunocompromised individuals, who develop severe interstitial pneumonia (PcP). However, it is known that asymptomatic or mild pulmonary infections, defined as colonization, are widely observed in the general adult population. So far, genetic and epidemiological data of P. jirovecii infections in Italy are rather scarce and limited to defined geographical regions, mainly regarding isolates from HIV-infected patients. The aim of this study was to evaluate the polymorphisms at the mtLSU-rRNA and the DHPS loci by the screening and genotyping of a cohort of patients from two major hospitals in Rome (Italy). The study included 263 patients divided into two groups, all enrolled consecutively from January 2006 to December 2010: (i) 38 immunocompromised subjects including 25 HIV-infected; (ii) 225 immunocompetent patients. Sixty-seven out of 263 patients (25.5%) were found positive after PCR amplification of the mtLSU-rRNA gene. Overall, genotyping at mtLSU-rRNA locus revealed that the genotype 2 was the most frequent. Sequences of the DHPS gene were obtained from 21 patients, 9 from immunocompromised patients (6 from HIV infected individuals), 12 from immunocompetent ones. Considering the most common DHPS mutations usually detected at amino acid positions 55 and 57 and potentially related to drug resistance, all samples analyzed showed the wild-type signatures. These are the first data in Italy on prevalence and genotypes of P. jirovecii regarding colonized immunocompetent adults. Further multicenter analyses on P. jirovecii infection will be necessary to better define the specific epidemiology of the disease in the Italian populations.

HIV-2 A-subtype gp125 C2-V3-C3 mutations and their association with CCR5 and CXCR4 tropism

The early events of the HIV replication cycle involve the interaction between viral envelope glycoproteins and their cellular CD4-chemokine (CCR5/CXCR4) receptor complex. In this study, for the first time, the HIV-2 A-subtype gp125C2-V3-C3 mutations and their tropism association were characterized by analyzing 149 HIV-2 sequences from the Los Alamos database. The analysis has strengthened the importance of C2-V3-C3 region as a determinant factor for co-receptor selection. Moreover, statistically significant correlations were observed between C2-V3-C3 mutations, and several correlated mutations were associated with CXCR4 and CCR5 co-receptor usage. A dendrogram showed two distinct clusters, with numerous associated mutations grouped, thus dividing CCR5- and CXCR4-tropic viruses. Fourteen X4-tropic virus mutations, all in V3 and C3 domains and forming highly significant subclusters, were found. Finally, R5 associations, two strong subclusters were observed, grouping several C2-V3-C3 mutated positions. These data indicate the possible contribution of C2-V3-C3 mutational patterns in regulating HIV-2 tropism.