Multiple Rounds Of Infection Research Articles

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

Lung-resident memory CD4 T cells do not divide extensively during protective responses against Influenza virus

Abstract Tissue-resident memory (TRM) CD4 T cells are emerging as critical orchestrators of immunity but little is known about their response kinetics during secondary challenges. We recently developed an adoptive transfer model in which virus-specific CD4 TRM isolated from lungs of Influenza A virus (IAV)-primed mice are transferred intranasally to new congenic hosts. Here, we track these donor TRM cells after IAV challenge of the adoptive hosts to gain insight into unique aspects of CD4 TRM responses to infection. Our findings demonstrate that the TRM rapidly activate elements of innate defense in the lung, including dendritic cells and NK cells. The TRM response is limited to the lung as no donor cells are detected in the spleen or mediastinal draining lymph nodes through the first week of infection. Although the CD4 TRM are highly protective and can clear even lethal IAV challenge in unprimed hosts, they undergo only limited division. To ask if the TRM-mediated protection requires influx of systemic T cells to the lung, we treated adoptive hosts with FTY720 to block the migration of host T cells during infection. TRM-mediated protection was not impacted by FTY720 treatment, indicating that the local memory CD4 cells can protect independently of T cell responses initiated in secondary lymphoid organs. Finally, we could detect the donor TRM in lungs for at least 21 days after infection and they were rapidly activated by antigen given intranasally. Our results thus show that lung CD4 TRM initiate highly protective responses in the lungs of otherwise naïve mice and have the capacity to respond against multiple rounds of infection. As such their generation through vaccination is a powerful way to improve immunity against seasonal and pandemic IAV.

High-Content Analyses of Vaccinia Plaque Formation.

Vaccinia virus plaque assays are employed for quantification of virus titer through serial dilution of virus on a monolayer of cells. Once the virus titer is diluted enough to allow for only few cells of the monolayer to be infected, clonal spread of infection can be detected by observing the lesion in the cell monolayer or using virus-specific staining methods. Beyond simple titration, plaque formation bares priceless underlying information about subtle virus-host interactions and their impact on virus spread during multiple rounds of infection. These include virus infectivity, mode of virus spread, virus replication rate, and spatiotemporal spread efficacy. How this underlying information can be harnessed using a high-content imaging setup is discussed here.

Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells.

The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

Antiviral Activity of Bictegravir and Cabotegravir against Integrase Inhibitor-Resistant SIVmac239 and HIV-1.

Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

BackgroundNewcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage.ResultsResults showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs.ConclusionsResults demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0659-3) contains supplementary material, which is available to authorized users.

Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells

The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection. We measured the kinetics of virus replication (VSV), antiviral paracrine signal upregulation and secretion, spatial spread of virus and paracrine antiviral signaling, and inhibition of virus production in antiviral-exposed A549 human lung epithelial cells. We found that initially infected cells released antiviral signals 4-to-7h following production of virus. However, the subsequent rapid dissemination of signal and fast induction of a robust and persistent antiviral state ultimately led to a suppression of infection spread. This work shows how cellular responses to infection and activation of antiviral responses can integrate to ultimately control infection spread across host cell populations.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

Dynamics of Mycobacteriophage-Mycobacterial Host Interaction: Evidence for Secondary Mechanisms for Host Lethality

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms.

Reaching for the Holy Grail: insights from infection/cure models on the prospects for vaccines for Trypanosoma cruzi infection.

Prevention of Trypanosoma cruzi infection in mammals likely depends on either prevention of the invading trypomastigotes from infecting host cells or the rapid recognition and killing of the newly infected cells by T. cruzi-specific T cells. We show here that multiple rounds of infection and cure (by drug therapy) fails to protect mice from reinfection, despite the generation of potent T cell responses. This disappointing result is similar to that obtained with many other vaccine protocols used in attempts to protect animals from T. cruzi infection. We have previously shown that immune recognition of T. cruzi infection is significantly delayed both at the systemic level and at the level of the infected host cell. The systemic delay appears to be the result of a stealth infection process that fails to trigger substantial innate recognition mechanisms while the delay at the cellular level is related to the immunodominance of highly variable gene family proteins, in particular those of the trans-sialidase family. Here we discuss how these previous studies and the new findings herein impact our thoughts on the potential of prophylactic vaccination to serve a productive role in the prevention of T. cruzi infection and Chagas disease.

Visualizing infection spread: Dual‐color fluorescent reporting of virus–host interactions

Although the molecular mechanisms by which host cells defend themselves against viral infection have been studied in great depth, and countermeasures viruses employ to suppress such defensive responses have been widely documented, relatively little attention has been devoted toward elucidating how such interactions between virus and host are resolved over multiple rounds of infection. Here, we describe the design, synthesis, and validation of a dual-color fluorescent reporter system to study how viral infections spread through a host cell monolayer and how the cellular innate immune system mounts an antiviral response. We employed recombinant, red fluorescent protein expressing mutants of a prototypical RNA virus, vesicular stomatitis virus to enable identification and tracking of infected cells. Further, we generated stable reporter cells that use green fluorescent protein to report on the expression of IFIT2, an interferon stimulated gene involved in the interference of viral protein translation, and a marker of antiviral defense activation. The presence of the fluorescent protein reporters had minimal effects on the normal behavior of the cells or viruses. Moreover, expression of the virus and cell reporters correlated with the kinetics of viral replication and activation of an anti-viral response, respectively. This two-color system enabled us to track and quantify in live cells how viral replication and activation of host defensive responses play out over multiple rounds of infection. Initial study of propagating infections demonstrated that antiviral activation over multiple rounds was critical for slowing and ultimately halting the spread of infection.

Gene Expression Profiling of Murine T-Cell Lymphoblastic Lymphoma Identifies Deregulation of S-Phase Initiating Genes.

AbstractAbstract 2395Human T-cell lymphoblastic lymphomas (T-LBLs) are neoplasms of immature T-cells and constitute a group of rare, heterogeneous and clinically very aggressive tumors. The molecular pathogenesis that contributes to T-LBL development is not fully elucidated. Since murine T-LBLs are histopathologically and phenotypically comparable to human T-LBLs, mouse models of T-LBLs are ideal to obtain additional insight into the mechanism of T-LBL development in humans. When injected into newborn mice of the NMRI inbred strain, the SL3-3 murine leukemia virus (MLV) induces various types of hematological malignancies, including T-LBLs. The oncogenic effects of the SL3-3 MLV are caused by integration of the viral genome into the host cell DNA through multiple rounds of infection, and subsequent deregulation of nearby cellular genes – a process defined as insertional mutagenesis. If the integration occurs near or within a gene of importance for cancer development, the cell in which the virus has integrated, may gain a growth advantage, eventually leading to malignant transformation and development of a full blown tumor. Screening the murine genome for resulting integration sites in the end-stage tumors, is therefore an efficient method for identifying genes involved in murine and potentially also human T-cell lymphomagenic processes.In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI inbred strain were inoculated with mutants of SL3-3 MLV. The mutants were affected in the glucocorticoid response element and an overlapping E-box of the viral enhancer in the long termial releat. By performing integration analysis on 19 and global gene expression profiling on 22 of the resulting T-LBL tumors, we determined both the effect of the retroviral integrations on the summarized expression of the nearby genes, and the deregulated pathways in the tumors. Fifty two different genes were identified within a 10 kb distance of the retroviral integrations, whereof 15 were specifically involved in G1/S phase transition. Gene expression dot-plots showed an activating effect of the retrovirus on Mr1, Stx6, Cask and Sh3gl3. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance (Mcm) and origin of recognition (Orc) pathway as well as downregulation in negative regulators of G1/S transition, indicating that murine T-LBLs have increased S-phase initiation. In conclusion, both the integration analysis and patterns of mRNA expression identified by gene expression profiling in the mouse models of T-LBL strongly indicate that genes involved in G1/S phase transition and/or S-phase initiation are deregulated suggesting similar mechanisms to be of importance in human T-LBL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.

Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

BackgroundThe identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle.ResultsHERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus.ConclusionsHERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.

Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy

Latency and ongoing replication have both been proposed to explain the drug-insensitive human immunodeficiency virus (HIV) reservoir maintained during antiretroviral therapy. Here we explore a novel mechanism for ongoing HIV replication in the face of antiretroviral drugs. We propose a model whereby multiple infections per cell lead to reduced sensitivity to drugs without requiring drug-resistant mutations, and experimentally validate the model using multiple infections per cell by cell-free HIV in the presence of the drug tenofovir. We then examine the drug sensitivity of cell-to-cell spread of HIV, a mode of HIV transmission that can lead to multiple infection events per target cell. Infections originating from cell-free virus decrease strongly in the presence of antiretrovirals tenofovir and efavirenz whereas infections involving cell-to-cell spread are markedly less sensitive to the drugs. The reduction in sensitivity is sufficient to keep multiple rounds of infection from terminating in the presence of drugs. We examine replication from cell-to-cell spread in the presence of clinical drug concentrations using a stochastic infection model and find that replication is intermittent, without substantial accumulation of mutations. If cell-to-cell spread has the same properties in vivo, it may have adverse consequences for the immune system, lead to therapy failure in individuals with risk factors, and potentially contribute to viral persistence and hence be a barrier to curing HIV infection.

Nef does not inhibit F-actin remodelling and HIV-1 cell–cell transmission at the T lymphocyte virological synapse

Nef, a HIV-1 pathogenesis factor, elevates virus replication in vivo and thus progression to AIDS by incompletely defined mechanisms. As one of its biological properties, Nef enhances the infectivity of cell-free HIV-1 particles in single round infections, however it fails to provide a significant and amplifying growth advantage for HIV-1 on such virus producing cells. A major difference between HIV-1 cell-free single round infections and virus replication kinetics on T lymphocytes consists in the predominant role of cell-associated virus transmission rather than cell-free infection during multiple round virus replication. HIV-1 cell-to-cell transmission occurs across close cell contacts also referred to as virological synapse (VS) and involves polarization of the F-actin cytoskeleton, formation of F-actin rich membrane bridges as well as virus budding to cell–cell contacts. Since Nef potently interferes with triggered actin remodelling in several cell systems to reduce e.g. cell motility and signal transduction, we set out here to address whether Nef also affects organization and possibly function of the T lymphocyte VS. We find that in addition to increasing infectivity of cell-free virions, Nef can also moderately enhance single rounds of HIV-1 cell–cell transmission between Jurkat T lymphocytes. This occurs without affecting cell conjugation efficiencies or polarization of F-actin and HIV-1 p24Gag at the VS, identifying actin remodelling at the VS as an example of Nef-insensitive host cell actin rearrangements. However, Nef-mediated enhancement of single round cell-free infection or cell-to-cell transmission does not potentiate over multiple rounds of infection. These results suggest that Nef affects cell-free and cell-associated HIV-1 infection by the same mechanism acting on the intrinsic infectivity of HIV-1 particles. They further indicate that the high efficacy of cell-to-cell transmission can compensate such infectivity defects. Nef therefore selectively interferes with actin remodelling processes involved in antiviral host cell defense while actin driven processes that promote virus propagation remain unaltered.

Analysis of the N-terminal region of the murine leukemia virus nucleocapsid protein

Lentiviruses such as the human immunodeficiency virus (HIV-1) and alpharetroviruses such as Rous Sarcoma virus encode an element that spans the precursor Gag (PrGag) protein capsid (CA) C-terminus, a spacer peptide (SP), and the N-terminus of nucleocapsid (NC). Perturbation of this element causes the assembly of aberrant, non-infectious virus particles. To determine whether this element is conserved in gammaretroviruses such as the Moloney murine leukemia virus (MLV), we examined the effects of insertion mutations in the N-terminal portion of the MLV NC coding region. Interestingly, we found that insertions of as many as twenty residues after the twelfth residue of MLV NC yielded proteins that directed the efficient assembly of virus particles. Virus morphologies and crosslink profiles appeared normal, and assembled viruses retained significant levels of infectivity in single cycle infection assays. Two variants were examined in the context of replicating virus constructs, and the mutations were found to be maintained during multiple rounds of infection in a cell culture system. These results suggest that the alpharetrovirus and lentivirus assembly elements either are not needed for gammaretroviruses, or are replaced by an alternative assembly element. Our results also indicate that the N-terminal region of MLV NC is amenable to genetic manipulation.

Replication of avian sarcoma virus in vivo requires an interaction between the viral RNA and the TpsiC loop of the tRNA(Trp) primer.

Reverse transcription in avian sarcoma virus (ASV) initiates from the 3' end of a tRNA(Trp) primer, which anneals near the 5' end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the T psi C loop of the tRNA(Trp) (U5-T psi C). We now demonstrate that this U5-T psi C interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-T psi C interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the T psi C loop of the tRNA(Trp). To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNA(Pro), while simultaneously randomizing the viral RNA U5-T psi C region. After five rounds of infection, the consensus sequence 5'-GPuPuCPy-3' emerged, which is complementary to the 5'-GGTTC-3' sequence found in the T psi C loop of tRNA(Pro). These observations confirm the importance of the U5-T psi C interaction in vivo.

Improving the display of proteins on filamentous phage

In phage display technology, polypeptides are displayed on the surface of filamentous bacteriophage by genetic fusion to a coat protein. However, the fraction of phage particles bearing the fusion protein can be low. Here we found that we could improve the display of a protein (Stoffel fragment of Taq polymerase fused to the p3 protein of the phage) by mutation of the signal sequence and use of helper phage with a protease-cleavable coat protein. Over multiple rounds of infection, proteolysis and binding to an anti- Taq antibody, we were able to select strongly for display of the fusion protein (>50-fold), and for mutations in the translation initiation region and in the signal sequence of the fusion. This suggests a general means of improving the display of proteins on phage.