Superinfection exclusion creates spatially distinct influenza virus populations.

Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing.

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

HIV Silencing and Inducibility Are Heterogeneous and Are Affected by Factors Intrinsic to the Virus.

Transcriptionally silent HIV proviruses form the major obstacle to eradicating HIV. Many studies of HIV latency have focused on the cellular mechanisms that maintain silencing of proviral DNA. Here we show that viral sequence variation affecting replicative ability leads to variable rates of silencing and ability to reactivate. We studied naturally occurring and engineered polymorphisms in a recently identified exonic splice enhancer (ESEtat) that regulates tat mRNA splicing and constructed viruses with increased (strain M1), reduced (strain M2), or completely absent (strain ERK) binding of splicing factors essential for optimal production of tat mRNA resulting in a corresponding change in Tat activity. The mutations affected viral replication, with M1 having wild-type (WT) kinetics, M2 exhibiting reduced kinetics, and ERK showing completely abrogated replication. Using single-round infection with green fluorescent protein (GFP)-expressing viruses to study proviral gene expression, we observed progressively greater rates of silencing relating to the degree of ESEtat disruption, with the WT strain at 53%, strain M2 at 69%, and strain ERK at 94%. By stimulating infected cells with a latency reversal agent (phorbol myristate acetate [PMA], panobinostat, or JQ1), we observed that the dose required to achieve 50% of the maximum signal was lowest in the WT, intermediate in M2, and highest in ERK, indicating progressively higher thresholds for reactivation. These results suggest that the ability of silent proviruses to reactivate from latency is variable and that minor differences in the viral sequence can alter the proportion of silenced viruses as well as the threshold required to induce silenced viruses to reactivate and express.IMPORTANCE A reservoir of infected cells in which the HIV genome is transcriptionally silent is acknowledged to be the principal barrier to eradicating the virus from an infected person. A number of cellular processes are implicated in this silencing; however, the viral factors that may contribute remain underexplored. Here we examined mutations altering the correct splicing of HIV gene products as a model to study whether differences in viral sequence can affect either the proportion of viruses that are active or silent or their ability to reactivate. We found that some naturally occurring variations result in viruses that are silenced at a higher rate and require a proportionally increased stimulus for reactivation from latency. These data suggest that the silencing and reactivation behavior of HIV exists in a spectrum, influenced by factors intrinsic to the virus.

TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.

No evidence of ongoing evolution in replication competent latent HIV-1 in a patient followed up for two years.

The persistence of infected T cells harbouring intact HIV proviruses is the barrier to the eradication of HIV. This reservoir is stable over long periods of time despite antiretroviral therapy. There has been controversy on whether low level viral replication is occurring at sanctuary sites periodically reseeding infected cells into the latent reservoir to account its durability. To study viral evolution in a physiologically relevant population of latent viruses, we repeatedly performed virus outgrowth assays on a stably treated HIV positive patient over two years and sequenced the reactivated latent viruses. We sought evidence of increasing sequence pairwise distances with time as evidence of ongoing viral replication. 64 reactivatable latent viral sequences were obtained over 103 weeks. We did not observe an increase in genetic distance of the sequences with the time elapsed between sampling. No evolution could be discerned in these reactivatable latent viruses. Thus, in this patient, the contribution of low-level replication to the maintenance of the latent reservoir detectable in the blood compartment is limited.