HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat

<p>Abstract</p> <p>Background</p> <p>Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance.</p> <p>Results</p> <p>Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all <it>in vitro </it>selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases.</p> <p>Conclusions</p> <p>These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.</p

Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

<p>Abstract</p> <p>Background</p> <p>Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2^431V, HXB2^436E+437T, HXB2^437Tand HXB2^437V).</p> <p>Results</p> <p>Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one.</p> <p>The observed decrease in RC for the double NC/p1 mutant (HXB2^436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during <it>in vitro </it>evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant.</p> <p>Conclusions</p> <p>The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.</p

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

BACKGROUND: HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. METHODS AND FINDINGS: We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. CONCLUSIONS: HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

We developed a rapid and sensitive method for the routine detection of all members of the enterovirus genus in different clinical specimens by using real-time TaqMan quantitative PCR. Multiple primer and probe sets were selected in the highly conserved 5′-untranslated region of the enterovirus genome. Our assay detected all 60 different enterovirus species tested, whereas no reactivity was observed with the viruses from the other genera of the picornaviridae family, e.g., hepatovirus and parechovirus. Weak cross-reactivity was observed with 7 of the 90 different high-titer rhinovirus stocks but not with rhinovirus-positive clinical isolates. Analysis of a well-characterized reference panel containing different enteroviruses at various concentrations demon-strated that the enterovirus real-time TaqMan PCR is as sensitive as most of the currently used molecular detection assays. Evaluation of clinical isolates demonstrated that the assay is more sensitive than the “gold standard” method, i.e., viral culture. Moreover, the PCR assay can be used on different clinical specimens, such as plasma, serum, nose and throat swabs, cerebrospinal fluid, and bronchoalveolar lavage, without apparent inhibition. Our data demonstrate that the real-time TaqMan PCR is a rapid and sensitive assay for the detection of enterovirus infection. The assay has a robust character and is easily standardized, which makes it an excellent alternative for the conventional time-consuming viral culture

Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region▿ †

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development

Shorter serial intervals in SARS-CoV-2 cases with Omicron BA.1 variant compared with Delta variant, the Netherlands, 13 to 26 December 2021

The SARS-CoV-2 Omicron variant has a growth advantage over the Delta variant because of higher transmissibility, immune evasion or shorter serial interval. Using S gene target failure (SGTF) as indication for Omicron BA.1, we identified 908 SGTF and 1,621 non-SGTF serial intervals in the same period. Within households, the mean serial interval for SGTF cases was 0.2-0.6 days shorter than for non-SGTF cases. This suggests that the growth advantage of Omicron is partly due to a shorter serial interval