Continued selection on cryptic SARS-CoV-2 observed in Missouri wastewater.

Deep sequencing of wastewater to detect SARS-CoV-2 has been used during the COVID-19 pandemic to monitor viral variants as they appear and circulate in communities. SARS-CoV-2 lineages of an unknown source that have not been detected in clinical samples, referred to as cryptic lineages, are sometimes repeatedly detected from specific locations. We have continued to detect one such lineage previously seen in a Missouri site. This cryptic lineage has continued to evolve, indicating continued selective pressure similar to that observed in Omicron lineages.

Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Tetherin inhibits HIV-1 release by directly tethering virions to cells.

Tetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. These findings allowed the design of a completely artificial protein, lacking sequence homology with native tetherin, that nevertheless mimicked its antiviral activity. We further show that tetherin is incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. These results indicate that tetherin functions autonomously and directly and that infiltration of virion envelopes by one or both of tetherin's membrane anchors is necessary, and likely sufficient, to tether enveloped virus particles that bud through the plasma membrane.

Diverse viral glycoproteins as well as CD4 co-package into the same human immunodeficiency virus (HIV-1) particles.

BACKGROUND: Retroviruses can acquire not only their own glycoproteins as they bud from the cellular membrane, but also some cellular and foreign viral glycoproteins. Many of these non-native glycoproteins are actively recruited to budding virions, particularly other viral glycoproteins. This observation suggests that there may be a conserved mechanism underlying the recruitment of glycoproteins into viruses. If a conserved mechanism is used, diverse glycoproteins should localize to a single budding retroviral particle. On the other hand, if viral glycoproteins have divergent mechanisms for recruitment, the different glycoproteins could segregate into different particles. RESULTS: To determine if co-packaging occurs among different glycoproteins, we designed an assay that combines virion antibody capture and a determination of infectivity based on a luciferase reporter. Virions were bound to a plate with an antibody against one glycoprotein, and then the infectivity was measured with cells that allow entry only with a second glycoprotein. We tested pairings of glycoproteins from HIV, murine leukemia virus (MLV), Rous sarcoma virus (RSV), vesicular stomatitis virus (VSV), and Ebola virus. The results showed that glycoproteins that were actively recruited into virions were co-packaged efficiently with each other. We also tested cellular proteins and found CD4 also had a similar correlation between active recruitment and efficient co-packaging, but other cellular proteins did not. CONCLUSION: Glycoproteins that are actively incorporated into HIV-1 virions are efficiently co-packaged into the same virus particles, suggesting that the same general mechanism for recruitment may act in many viruses.

Retrovirus glycoprotein functionality requires proper alignment of the ectodomain and the membrane-proximal cytoplasmic tail.

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the -1L, -2L, -3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.

Multiple Gag domains contribute to selective recruitment of murine leukemia virus (MLV) Env to MLV virions.

Retroviruses, like all enveloped viruses, must incorporate viral glycoproteins to form infectious particles. Interactions between the glycoprotein cytoplasmic tail and the matrix domain of Gag are thought to direct recruitment of glycoproteins to native virions for many retroviruses. However, retroviruses can also incorporate glycoproteins from other viruses to form infectious virions known as pseudotyped particles. The glycoprotein murine leukemia virus (MLV) Env can readily form pseudotyped particles with many retroviruses, suggesting a generic mechanism for recruitment. Here, we sought to identify which components of Gag, particularly the matrix domain, contribute to recruitment of MLV Env into retroviral particles. Unexpectedly, we discovered that the matrix domain of HIV-1 Gag is dispensable for generic recruitment, since it could be replaced with a nonviral membrane-binding domain without blocking active incorporation of MLV Env into HIV virions. However, MLV Env preferentially assembles with MLV virions. When MLV and HIV particles are produced from the same cell, MLV Env is packaged almost exclusively by MLV particles, thus preventing incorporation into HIV particles. Surprisingly, the matrix domain of MLV Gag is not required for this selectivity, since MLV Gag containing the matrix domain from HIV is still able to outcompete HIV particles for MLV Env. Although MLV Gag is sufficient for selective incorporation to occur, no single Gag domain dictates the selectivity. Our findings indicate that Env recruitment is more complex than previously believed and that Gag assembly/budding sites have fundamental properties that affect glycoprotein incorporation.

Genome-wide CRISPR/Cas9 screen reveals JunB downmodulation of HIV co-receptor CXCR4.

HIV-1 relies extensively on host cell machinery for replication. Identification and characterization of these host-virus interactions is vital to our understanding of viral replication and the consequences of infection in cells. Several prior screens have identified host factors important for HIV replication but with limited replication of findings, likely due to differences in experimental design and conditions. Thus, unidentified factors likely exist. To identify novel host factors required for HIV-1 infection, we performed a genome-wide CRISPR/Cas9 screen using HIV-induced cell death as a partitioning method. We created a gene knockout library in TZM-GFP reporter cells using GeCKOv2, which targets 19,050 genes, and infected the library with a lethal dose of HIV-1NL4-3. We hypothesized that cells with a knockout of a gene critical for HIV infection would survive while cells with a knockout of a non-consequential gene would undergo HIV-induced death and be lost from the population. Surviving cells were analyzed by high throughput sequencing of the integrated CRISPR/Cas9 cassette to identify the gene knockout. Of the gene targets, an overwhelming majority of the surviving cells harbored the guide sequence for the AP-1 transcription factor family protein, JunB. Upon the generation of a clonal JunB knockout cell line, we found that HIV-1NL4-3 infection was blocked in the absence of JunB. The phenotype resulted from downregulation of CXCR4, as infection levels were recovered by reintroduction of CXCR4 in JunB KO cells. Thus, JunB downmodulates CXCR4 expression in TZM-GFP cells, reducing CXCR4-tropic HIV infection.

Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage.

BACKGROUND: The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. METHODS: We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. FINDINGS: We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7 × 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13 consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. INTERPRETATION: We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. FUNDING: The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings. We detected 106 samples that were positive for SARS-CoV-2 viral RNA, demonstrating that SARS-CoV-2 can be detected in continuous air samples collected from a variety of real-world settings. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air sampling is a scalable, high throughput surveillance tool that could be used in conjunction with other methods for detecting respiratory pathogens in congregate settings.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings and detected 106 SARS-CoV-2 positive samples, demonstrating SARS-CoV-2 can be detected in air collected from daily and weekly sampling intervals. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection in the community. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air surveillance is a scalable, cost-effective, and high throughput alternative to individual testing for detecting respiratory pathogens in congregate settings.

Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner.

Sequencing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from wastewater has become a useful tool in monitoring the spread of viral variants. Approaches to this task have been varied, relying on differing sequencing methods and computational analyses. We used a novel computation workflow based on amplicon sequencing of SARS-CoV-2 spike domains in order to track viral populations in wastewater. As part of this workflow, we developed a program, SAM Refiner, that has a variety of outputs, including novel variant reporting as well as functions designed to remove polymerase chain reaction (PCR) generated chimeric sequences. With these methods, we were able to track viral population dynamics over time. We report here on the emergence of two variants of concern, B.1.1.7 (Alpha) and P.1 (Gamma), and their displacement of the D614G B.1 variant in a Missouri sewershed.

ßTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin.

The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein ßTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the ßTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both ßTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/ßTrCP ubiquitin ligase.

Characterization of mre11 loss following HSV-1 infection.

Herpes simplex virus induces the activation of the cellular DNA double strand break response pathway dependent upon initiation of viral DNA replication. The MRN complex, consisting of Mre11, Rad50 and Nbs1, is an essential component of the DNA double strand break response and other reports have documented its presence at sites of viral DNA replication, interaction with ICP8 and its contribution to efficient viral DNA replication. During our characterization of the DSB response following infection of normal human fibroblasts and telomerase-immortalized keratinocytes, we observed the loss of Mre11 protein at late times following infection. The loss was not dependent upon ICP0, the proteasome or lysosomal protease activity. Like activation of the DSB response pathway, Mre11 loss was prevented under conditions which inhibited viral DNA replication. Analysis of a series of mutant viruses with defects in cleavage and packaging (UL6, UL15, UL17, UL25, UL28, UL32) of viral DNA or in the maturational protease (UL26) failed to identify a viral gene product necessary for Mre11 loss. Inactivation of ATM, a key effector kinase in the DNA double strand break response, had no effect on Mre11 loss and only a moderate effect on HSV yield. Finally, treatment of uninfected cells with the topoisomerase I inhibitor camptothecin, to induce generation of free DNA ends, also resulted in Mre11 loss. These results suggest that Mre11 loss following infection is caused by the generation of free DNA ends during or following viral DNA replication.