Search

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic caused by it, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been undergoing a genetic diversification leading to the emergence of new variants. Nevertheless, a clear definition of the genetic signatures underlying the circulating variants is still missing. Here, we provide a comprehensive insight into mutational profiles characterizing each SARS-CoV-2 variant, focusing on spike mutations known to modulate viral infectivity and/or antigenicity. We focused on variants and on specific relevant mutations reported by GISAID, Nextstrain, Outbreak.info, Pango, and Stanford database websites that were associated with any clinical/diagnostic impact, according to published manuscripts. Furthermore, 1,223,338 full-length high-quality SARS-CoV-2 genome sequences were retrieved from GISAID and used to accurately define the specific mutational patterns in each variant. Finally, mutations were mapped on the three-dimensional structure of the SARS-CoV-2 spike protein to assess their localization in the different spike domains. Overall, this review sheds light and assists in defining the genetic signatures characterizing the currently circulating variants and their clinical relevance. IMPORTANCE Since the emergence of SARS-CoV-2, several recurrent mutations, particularly in the spike protein, arose during human-to-human transmission or spillover events between humans and animals, generating distinct worrisome variants of concern (VOCs) or of interest (VOIs), designated as such due to their clinical and diagnostic impacts. Characterizing these variants and their related mutations is important in tracking SAR-CoV-2 evolution and understanding the efficacy of vaccines and therapeutics based on monoclonal antibodies, convalescent-phase sera, and direct antivirals. Our study provides a comprehensive survey of the mutational profiles characterizing the important SARS-CoV-2 variants, focusing on spike mutations and highlighting other protein mutations.

We describe the establishment of a seronegative occult hepatitis B virus (HBV) infection (OBI) in a successfully vaccinated infant who underwent liver transplantation from an donor positive for antibody to hepatitis B core antigen (anti-HBc). The use of highly sensitive droplet digital polymerase chain reaction assays revealed a not negligible and transcriptionally active intrahepatic HBV reservoir (circular covalently closed DNA, relaxed circular DNA, and pregenomic RNA: 5.6, 2.4, and 1.1 copies/1000 cells, respectively), capable to sustain ongoing viral production and initial liver damage. Next-generation sequencing revealed a peculiar enrichment of hepatitis B surface antigen vaccine-escape mutations that could have played a crucial role in OBI transmission. This clinical case highlights the pathobiological complexity and the diagnostic challenges underlying OBI., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Although several cases of hepatitis B virus reactivation have been described in patients with a history of hepatitis B virus infection while undergoing treatment for hepatitis C virus infection with direct acting antivirals, the question of whether hepatitis B virus surface antigen immune-escape mutations might play a role has not been addressed so far., Case Presentation: We report a case of hepatitis B virus reactivation in a Caucasian patient infected with hepatitis C virus during treatment with sofosbuvir and velpatasvir. A 50-year-old man with a genotype 1a hepatitis C virus infection was considered for therapy. His serological profile was hepatitis B virus surface antigen-negative, hepatitis B virus core antibody-positive, hepatitis B virus surface antibody-negative, and anti-hepatitis D virus-positive. The detection of hepatitis B virus deoxyribonucleic acid (DNA) indicated active viral replication during the direct acting antiviral treatment that spontaneously returned to undetectable levels after treatment completion. Starting from week 12 after the end of treatment, hepatitis B virus surface antibody titers and hepatitis B virus e antibody developed. Sequencing analysis revealed the hepatitis B virus genotype D3 and the presence of two relevant immune-escape mutations (P120S and T126I) in the major hydrophilic region by analyzing the S region., Conclusions: We speculate that the presence of the hepatitis B virus surface antigen mutations, endowed with the enhanced capability to elude the immune response, could play a role in hepatitis B virus reactivation. This observation confirms that occult hepatitis B infection should also be carefully monitored, through surveillance of the hepatitis B virus viral load before and during direct acting antiviral treatment of hepatitis C virus.

The aim of this study is to evaluate the amino acid variability of HIV-1 Gp41, C2-V3, and Nef in a group of patients characterized by different disease progression rates. HIV-1 sequences were collected from 19 Long term non progressor patients (LTNPs), 9 slow progressors (SPs), and 11 rapid progressors (RPs). Phylogenetic trees were estimated by MEGA 6. Differences in amino acid variability among sequences belonging to the 3 groups have been evaluated by amino acid divergence, Shannon entropy analysis, and the number of amino acid mutations (defined as amino acid variations compared with HxB2). The involvement of amino acid mutations on epitope rich regions was also investigated. The population was mainly composed of males (74.3%) and HIV-1 subtype B strains (B: 92.32%, CRF&#95;12BF, A1, C: 2.56% each). Viral load (log 10 copies/mL) and CD4 + T cell count (cells/mm 3 ) were 3.9 (3.5-4.2) and 618 (504-857) in LTNPs, 3.3 (2.8-4.7) and 463 (333-627) in SPs, and 4.6 (4.3-5.3) and 201 (110-254) in RPs. Gp41 and C2-V3 amino acid divergence was lower in LTNP and SP strains compared to RPs (median value: 0.085 and 0.091 vs. 0.114, p = 0.005 and 0.042) and a trend of lower variability was observed for Nef (p = 0.198). A lower entropy value was observed at 10, 3, and 7 positions of Gp41, C2-V3, and Nef belonging to LTNPs and at 7, 3, and 1 positions of Gp41, C2-V3, and Nef belonging to SPs compared with RPs (p < 0.05). Focusing on epitope rich regions, again a higher degree of conservation was observed in Gp41 and C2-V3 sequences belonging to LTNPs and SPs compared to those belonging to RPs. This study shows that the extent of amino acid variability correlates with a different HIV-1 progression rate. This variability also involves CTL epitope rich regions, thus suggesting its involvement in the immune escape process modulation.

Background: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe., Methods: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence., Results: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties., Conclusions: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

Background: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation., Methods: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry., Results: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001)., Conclusions: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification., Methods: 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay., Results: Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate., Conclusions: Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

While the selection of complex HBV drug-resistance patterns on therapeutic failure can compromise the efficacy of anti-HBV therapies, recent data show that patients failing treatment without drug-resistance have a rate of virological success close to drug-naive patients. The goal of this study is defining, in clinical practice, the burden of drug-resistance mutations in a cohort of patients treated with anti-HBV drugs. Prevalence and patterns of drug-resistance were analyzed by RT-sequencing in 204 patients infected chronically: 148 experiencing virological rebound (defined as an increase in serum HBV-DNA > 20 IU/ml after achieving virological success [HBV-DNA < 20 IU/ml]), and 56 null/partial responders (always detectable serum HBV-DNA [>20 IU/ml] within 48 weeks of therapy). The highest rate of drug-resistance was observed in patients experiencing virological rebound (prevalence, 79.1%). Conversely, almost half (46.4%) null/partial responders have no evidence of drug-resistance. The rate of drug-resistance was higher in patients treated with lamivudine (76.8% [109/142]) and telbivudine (83.3% [5/6]), followed by adefovir (62.5% [15/24]), and entecavir (52.2% [12/23]). Complex mutational patterns characterized by the co-presence of rtM204V/I-rtA181T/V (impairing the efficacy of all anti-HBV drugs) were detected in four patients (2.7%) with virological rebound. Drug-resistance is the main cause of failure to therapy in patients experiencing virological rebound, supporting the need of rapid switch to anti-HBV drugs with higher genetic barrier and potency (entecavir/tenofovir). Conversely, nearly half of null/partial responders shows no evidence of drug-resistance mutations, maintaining high chance of achieving therapeutic success with the same class of drug. In this setting, genotypic resistance may help in selecting patients still carrying wild-type viruses, that may take major benefits from antiviral treatment., (Copyright © 2013 Wiley Periodicals, Inc.)

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 gp125(C2-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.

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 (E57A(rev)) and N86S(rev) with the ENF resistance gp41 mutations Q40H (Q40H(gp41)) and L45M(gp41). In addition, the presence at week 48 of the E57A(rev) 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 Q40(gp41) and L45(gp41) 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 Q40H(gp41) and L45M(gp41) 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.

During its spread among humans, HIV-1 has developed an extraordinary degree of genetic diversity. The pol region encoding for viral enzymes such as the reverse transcriptase and the protease, and the env region encoding for the viral glycoprotein gp41 are subjected not only to natural variation, but also to the selection pressure imposed by the pharmacologic treatment. Under these conditions in HIV-1 infected people, the virus is able to escape from antiviral drugs by accumulating mutations, either alone or in clusters. The patterns of mutations accumulated by HIV-1 under drug pressure are quite variable, depending on the backbone of virus strains, the level and type of pharmacologic pressure, and the length of therapy. To date, a high number of mutations in protease, reverse transcriptase, and gp41 have been associated with reduced susceptibility to the antiretroviral drugs currently available. However, a number of studies continuously highlight the existence of additional mutations beyond those currently known to be involved in the development of drug resistance in vivo. Most of these so-called "novel" mutations are involved in agonistic correlations with the classical drug resistance mutations on divergent evolutionary pathways, and are associated with an increased resistance to specific drugs. At the same time, the presence of some novel mutations at therapeutic failure has also been significantly associated with an increase of viremia, thus suggesting that they may also play a compensatory role leading to improved viral replication. Interestingly, some natural polymorphisms in drug-naive patients have been significantly associated with the development of drug resistance mutations at failure, thus suggesting their ability to decrease the genetic barrier to the development of drug resistance. In contrast, other novel mutations are negatively associated with specific antiviral treatment, showing negative interactions with relevant drug resistance mutations, and are associated with increased susceptibility to specific drugs. This article reviews the importance of recognition and the clinical relevance of novel mutations involved in resistance to the currently used antiretroviral drugs, discussing in particular the role of novel drug resistance mutations in the reverse transcriptase enzyme. Such novel mutations should be considered for improved prediction of clinical response to antiretroviral drugs and for assessing the efficacy of next-generation drugs.

Objectives: To investigate gp41 variability and correlation with viro-immunological parameters in 54 HIV-1-infected patients receiving enfuvirtide added as single active drug to a failing regimen., Methods: One hundred and two HIV-1 gp41 sequences and clinical follow-up from 54 enfuvirtide-treated patients were analysed from baseline to week 36 of treatment. The association of mutations with viraemia/CD4 count was assessed by Mann-Whitney test., Results: The addition of enfuvirtide to the failing regimen induced at week 4 a viraemia decrease from 5.1 to 4.3 log(10)/mL (P = 0.0002) and a CD4 increase from 48 to 106 cells/mm(3) (P = 0.008). While viraemia rebounded to 4.8 and 4.6 log(10)/mL at week 12 and 36, respectively, CD4 continued to increase to 136 cells/mm(3) at week 36. Enfuvirtide resistance mutations, rarely found at baseline, occurred in 45/54 (83.3%) enfuvirtide-treated patients. V38A/E were the most represented mutations at all time-points. The presence of V38A/E was significantly associated with a 4.5-fold CD4 increase from baseline to week 24 and with a 6-fold increase at week 36 (P = 0.004 and 0.02 compared without V38A/E, respectively), without significant correlation with viraemia. In contrast, Q40H + L45M (present in six enfuvirtide-treated patients at week 36) correlated with CD4 loss from baseline to week 36 (P = 0.02), without significant correlation with viraemia. Mutation N126K (observed in six enfuvirtide-treated patients, never found at baseline) abrogates the fourth gp41 glycosylation site and correlates with a 2.1-fold CD4 increase at week 24., Conclusions: Specific enfuvirtide resistance mutations (V38A/E) are associated with a sustained CD4 increase, without remarkable effects upon viraemia. This CD4 recovery, due to virus- and immune-mediated mechanisms most probably not applicable to protease/reverse transcriptase inhibitors, is important for innovative therapeutic strategies.

The aim of this study was to evaluate the prevalence of genotypic resistance for each drug-class, and of single resistance-mutations in 1075 HIV-1 infected multi-treated patients undergoing their first genotypic resistance-test (GRT) after virological failure, over the years 1999-2003. First GRT was requested in 2003 for patients at earlier stages of failure, with less advanced disease, higher CD4-cell-count, lower HIV-RNA, and lower drug-experience with respect to 1999. Prevalence of resistance to all three drug-classes decreased from 33.3% in 1999 to 14.8% in 2003 (p < 0.001). Patients with protease-inhibitor (PI) resistant viruses decreased from 68.1% in 1999 to 34.1% in 2003 (p < 0.001); patients with nucleoside reverse transcriptase-inhibitor (NRTI) resistant viruses remained unchanged (85.4% in 1999; 86.4% in 2003); patients with non-NRTI (NNRTI) resistant viruses increased from 36.1% in 1999 to 52.3% in 2003 (p = 0.005) (corresponding to an increased NNRTI-use and decreased PI-use). From 1999 to 2003, resistance-mutations to drugs with high genetic-barrier significantly decreased (L90M/V82A/M46I/I54V/G73S/I84V/G48V for PIs; M41L/D67N/L210W/V1181 for NRTIs, p < 0.05), while mutations to drugs with low genetic-barrier increased (D30N in protease, M184V/K103N/V108I in reverse transcriptase, p < 0.05). Taken together, earlier recruitment to first GRT in patients with less severe disease, and with lower prevalence of drug-resistant viruses may further improve therapeutic strategies aimed at longer and greater control of HIV-related disease progression.

Phylogenetic analysis and evaluation of drug-resistance were carried out upon 59 plasma samples from 58 treatment-naïve HIV-1 infected patients from Mozambique, enrolled in a free antiviral-therapy protocol in the frame of Drug-Resource-Enhancement against AIDS and Malnutrition (DREAM) programme. Sequencing of the first 1,300 bases of the pol-gene shows that all virus strains cluster within clade C, with the exception of a single patient carrying a G-subtype virus. Relevant mutations in the reverse transcriptase (RT) are rare: 118A/I/L/G (four patients), 179E/D/I (three patients), 333E/D (two patients), 101R, and 210F (one patient each). In Protease (PR), V82I (10.3%) is the only relevant mutation, while natural polymorphisms/secondary mutations are found, some at very high frequency: 20R (25.9%), 36I (91.4%), 36L (8.6%), 60E (31.0%), 63P (29.3%), and 93L (96.6%). Among them, mutations with a frequency >25% were further investigated to assess their covariation pattern with PI resistance associated mutations. The pattern of covariation observed for K20R and D60E (but not L63P and M36I) was different between C and B subtype isolates from PR-inhibitor-treated patients. The sequences were also analyzed to calculate the ratio of non-synonymous to synonymous substitution. The ratio for PR and RT was 0.116 and 0.093, respectively, suggesting a greater conservation in RT than PR in both subtypes B and C HIV strains. Taken together, the results demonstrate a consistent clade-homogeneity of viral strains circulating in Mozambique, and the very limited presence, in drug-naïve patients, of mutations associated with resistance to RT-inhibitors. The high frequency of secondary mutations/polymorphisms in HIV-PR deserves further studies to evaluate its relevance in clinical settings., ((c) 2005 Wiley-Liss, Inc.)

To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naive patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 A of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naive patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.