We have previously shown that HIV-1 superinfected Zambian seroconverters mount low binding and neutralizing antibody responses to their primary HIV-1 infecting virus, which could increase susceptibility to re-infection. Here, we investigated if antibody-dependent cellular cytotoxicity (ADCC), a process by which virus-infected cells are killed, was also reduced. Superinfected individuals exhibited low ADCC activity compared to non-superinfected individuals, but similar levels of CMV-reactive binding antibodies, suggesting superinfected individuals are capable of generating and maintaining virus-specific antibodies.
HIV-1 superinfection; ADCC; HIV-1 ADCC; HIV dual infection
One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development.
Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clade A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70–80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV-1 directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV-1 infection. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV-1 infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.
Replication competent adenovirus (RC-Ad) vectors mediate robust transgene expression by virtue of amplifying transgenes by replication, but also put patients at a risk of frank adenovirus infection. In contrast, E1-deleted replication defective Ad (RD-Ad) vectors are safer, but produce substantially less transgene product. To generate a robust, but safer adenoviral vector, we created a “single cycle" adenovirus (SC-Ad) vector that replicates its genome and transgene, but that does not cause adenovirus infections by deleting the capsid cement protein IIIa in low seroprevalence adenovirus serotype 6. Ad6-ΔIIIa can be produced in IIIa-expressing cell lines. In normal cells, Ad6-ΔIIIa replicates its genome and transgene, but fails to package its DNA or form mature virus. SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad in human and mouse cells in vitro and in vivo in mice. These data suggest that SC-Ads may be safer amplifying vectors for vaccine and therapeutic applications.
adenovirus; replication-competent; replication-defective; single-cycle; amplification; vaccines; gene therapy
Cardioviruses of the Encephalomyocarditis virus (EMCV) and Theilovirus species, encode small, amino-terminal proteins called Leaders (L). Phosphorylation of the EMCV L (LE) at two distinct sites by CK2 and Syk kinases is important for virus-induced Nup phosphorylation and nucleocytoplasmic trafficking inhibition. Despite similar biological activities, the LE phosphorylation sites are not conserved in the Theiloviruses, Saffold virus (LS, SafV) or Theiler’s murine encephalitis virus (LT, TMEV) sequences even though these proteins also become phosphorylated in cells and cell-free extracts. Site prediction algorithms, combined with panels of site-specific protein mutations now identify analogous, but not homologous phosphorylation sites in the Ser/Thr and Theilo protein domains of LT and LS, respectively. In both cases, recombinant AMP-activated kinase (AMPK) was reactive with the proteins at these sites, and also with LE, modifying the same residue recognized by CK2.
Cardiovirus; Leader protein; phosphorylation; AMPK
Respiratory syncytial virus RNA dependent RNA polymerase (RdRp) initiates RNA synthesis from the leader (le) and trailer-complement (trc) promoters. The RdRp can also add nucleotides to the 3′ end of the trc promoter by back-priming, but there is no evidence this occurs at the le promoter in infected cells. We examined how environmental factors and RNA sequence affect de novo RNA synthesis versus back-priming using an in vitro assay. We found that replacing Mg2+ with Mn2+ in the reaction buffer increased de novo initiation relative to back-priming, and different lengths of trc sequence were required for the two activities. Experiments with le RNA showed that back-priming occurred with this sequence in vitro, but less efficiently than with trc RNA. These findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay.
Respiratory syncytial virus; paramyxovirus; mononegavirales; RNA dependent RNA polymerase; back-priming; RNA synthesis
The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export.
HIV-1; Env; Gag; RNA export; RRE
Antibody B4e8 exhibits modest cross-neutralizing activity, with preference for HIV subtype B. This preference might be explained by B4e8’s extensive interaction with Arg315, which occurs at the center of most subtype B V3 sequences but is replaced by Gln in subtype C. The extent to which B4e8’s ability to neutralize subtype C strains is hindered by Gln315 and/or other factors, e.g. epitope masking, is unclear. We confirmed here that an Arg315-to-Gln substitution in a subtype B virus abrogates B4e8 neutralizing activity. Conversely, B4e8-resistant subtype C viruses were rendered sensitive upon Gln-to-Arg substitution. V2 region swapping between B4e8-sensitive and- resistant subtype C strains revealed a role for V2 in limiting B4e8 access, but this was less significant than the absence of Arg315. Our findings, while illustrating the importance of Arg315 for B4e8, suggest that some subtype C strains may be vulnerable to B4e8 derivatives capable of binding stronger to Gln315-containing sequences.
Neutralizing antibody; Arg315; V3 masking; V2 region; angle of interaction; subtype C
The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant.
simian hemorrhagic fever virus; subgenomic mRNAs; minor structural proteins; infectious clone
The response to influenza virus (IAV) infection and severity of disease is highly
variable in humans. We hypothesized that one factor contributing to this variability is
the presence of specific respiratory tract (RT) microbes. One such microbe is
Streptococcus pneumoniae (Sp) that is carried asymptomatically in the RT
of many humans. In a mouse co-infection model we found that in contrast to secondary
bacterial infection that exacerbates disease, Sp colonization 10 days
prior to IAV protects from virus-induced morbidity and lung pathology. Using mutant
Sp strains, we identified a critical role for the bacterial virulence
factor pneumolysin (PLY) in mediating this protection. Colonization with the
PLY-sufficient Sp strain induces expression of the immune-suppressive
enzyme arginase 1 in alveolar macrophages (aMø) and correlates with attenuated
recruitment and function of pulmonary inflammatory cells. Our study demonstrates a novel
role for PLY in Sp-mediated protection by maintaining aMø as
"gatekeepers" against virus-induced immunopathology.
influenza virus; Streptococcus pneumoniae; pneumolysin; respiratory tract co-infection; immunopathology; alveolar macrophages; inflammatory monocytes; arginase I; iNOS; protection
Granzyme B (GrzB) is expressed by activated T cells and mediates cellular apoptosis. GrzB also acts as an extracellular protease involved in tissue degradation. We hypothesized that GrzB production from activated memory CD4 T cells may be associated with HIV pathogenesis. We found that stimulated memory CD4 T cells (via costimulation, cytokines, and TLR ligands) concomitantly produced GrzB and HIV. Both GrzB and HIV expression were mainly restricted to CCR5-expressing memory CD4+CD45RO+ T cells, including Th1 and Th17 subsets. Activated memory CD4 T cells also mediated tissue damage, such as disruption of intestinal epithelial monolayers. In non-human primates, CD4 T cells of rhesus macaques (pathogenic SIV hosts) expressed higher GrzB compared to African green monkeys (non-pathogenic SIV hosts). These results suggest that GrzB from CCR5+ memory CD4 T cells may have a role in cellular and tissue pathologies during HIV infection.
CCR5; Granzyme B; HIV replication; Enteropathy; Memory CD4 T cells; SIV pathogenesis
Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection.
•Transmitted/founder viruses are known to be relatively interferon resistant.•Transmitted/founder HIV-1 viruses have fully functional accessory genes Vpu, Vif and Nef.•Vpu, Vif and Nef functions do not appear to differ between acute and chronic infection.
HIV; Vpu; Nef; Vif; Accessory; Acute; Chronic
The 2014 epidemic of Ebola virus disease (EVD) has had a devastating impact in West Africa. Sequencing of ebolavirus (EBOV) from infected individuals has revealed extensive genetic variation, leading to speculation that the virus may be adapting to humans, accounting for the scale of the 2014 outbreak. We computationally analyze the variation associated with all EVD outbreaks, and find none of the amino acid replacements lead to identifiable functional changes. These changes have minimal effect on protein structure, being neither stabilizing nor destabilizing, are not found in regions of the proteins associated with known functions and tend to cluster in poorly constrained regions of proteins, specifically intrinsically disordered regions. We find no evidence that the difference between the current and previous outbreaks is due to evolutionary changes associated with transmission to humans. Instead, epidemiological factors are likely to be responsible for the unprecedented spread of EVD.
•We study the current and previous outbreaks of Ebola virus disease.•There are many non-synonymous (amino-acid altering) changes in the viral sequences.•We can identify no changes that alter any molecular functions in the virus.•The large number of infections is probably due to epidemiological factors.
Ebola; Evolution; Adaptation; Protein structure; Protein function
Despite mounting evidence of the high disease burden of influenza in tropical regions, relatively little viral sequence data is available from tropical countries in the Western hemisphere. To understand the evolutionary dynamics of influenza A and B viruses in Managua, Nicaragua, we performed a phylogenetic analysis of 1,956 influenza viruses, including 335 collected for this study during 2007–2010 from a population-based cohort in Managua. North America was consistently identified as the most significant source of influenza virus diversity in Managua, although South America and Mexico were important viral sources during the 2009 A/H1N1 pandemic. The low number of viral introductions of Central American origin may reflect differences in the seasonality of influenza in Nicaragua versus neighboring countries, and underscores the need for additional data in this understudied region.
influenza; evolution; Central America; molecular epidemiology
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.
Jaagsiekte sheep retrovirus; Env; Transformation; Type II pneumocyte
Analysis of the herpes simplex virus-1 (HSV-1) genome reveals two candidate p53 responsive elements (p53RE), located in proximity to the replication origins oriL and oriS, referred to as p53RE-L and p53RE-S, respectively. The sequences of p53RE-L and p53RE-S conform to the p53 consensus site and are present in HSV-1 strains KOS, 17, and F. p53 binds to both elements in vitro and in virus-infected cells. Both p53RE-L and p53RE-S are capable of conferring p53-dependent transcriptional activation onto a heterologous reporter gene. Importantly, expression of the essential immediate early viral transactivator ICP4 and the essential DNA replication protein ICP8, that are adjacent to p53RE-S and p53RE-L, are repressed in a p53-dependent manner. Taken together, this study identifies two novel functional p53RE in the HSV-1 genome and suggests a complex mechanism of viral gene regulation by p53 which may determine progression of the lytic viral replication cycle or the establishment of latency.
Herpes simplex virus 1; p53; responsive elements; gene expression
Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway.
HSV-1; nuclear envelope; fusion; UL34; gB; gH; US3
The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.
SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition.
SAMHD1; SNPs; HIV-1; Vpx; dNTPs; LINE-1
The Autographa californica multiple nucleopolyhedrovirus ac92 is a conserved baculovirus gene with homology to flavin adenine dinucleotide-linked sulfhydryl oxidases. Its product, Ac92, is a functional sulfhydryl oxidase. Deletion of ac92 results in almost negligible levels of budded virus (BV) production, defects in occlusion-derived virus (ODV) co-envelopment and their inefficient incorporation into occlusion bodies. To determine the role of sulfhydryl oxidation in the production of BV, envelopment of nucleocapsids, and nucleocapsid incorporation into occlusion bodies, the Trichoplusia ni single nucleopolyhedrovirus ortholog, Tn79, was substituted for ac92. Tn79 was found to be an active sulfhydryl oxidase that substituted for Ac92, resulting in the production of infectious BV, albeit about 10-fold less than an ac92-containing virus. Tn79 rescued defects in ODV morphogenesis caused by a lack of ac92. Active Tn79 sulfhydryl oxidase activity is required for efficient BV production, ODV envelopment, and their subsequent incorporation into occlusion bodies in the absence of ac92.
We evaluated the replication efficiency of the HIV reverse transcriptase (RT) mutants K103N, G190A, and G190S, which confer resistance to the non-nucleoside RT inhibitor efavirenz, using growth competition assays in cell culture. In the absence of efavirenz, the fitness hierarchy was G190S < G190A < K103N < wild-type. The fitness reduction of G190S relative to K103N was less evident at high efavirenz concentrations, although K103N still replicated more efficiently. Efficiency of RNase H cleavage and RNA-dependent DNA synthesis from tRNALys,3 correlated with relative fitness, in biochemical studies of mutant RTs. Presteady state and steady state polymerization assays using DNA primers detected no abnormalities. This work is consistent with previous studies demonstrating that initiation of viral DNA synthesis is reduced in mutants with slowed RNase H cleavage, and suggests that both abnormalities contribute to the replication defect of these mutants. It also suggests that high concentrations of efavirenz are unlikely to favor the selection of G190S clinically.
HIV-1 drug resistance; Replication fitness; RNase H cleavage; tRNALys; 3 priming; Efavirenz; G190S
Efavirenz resistance during HIV-1 treatment failure is usually associated with the reverse transcriptase mutation K103N. L100I, V108I, or P225H can emerge after K103N and increase its level of efavirenz resistance. K103N + L100I is the most drug-resistant of the double mutants but is the least common clinically. We hypothesized that differences in replication efficiency, or fitness, influence the relative frequencies of these secondary efavirenz resistance mutations in clinical isolates. We measured fitness of each secondary mutant introduced into HIVNL4-3, alone and in combination with K103N, using growth competition assays in H9 cells. In the absence of efavirenz, the fitness of V108I was indistinguishable from wild type. K103N, L100I, and P225H were minimally, but consistently, less fit than wild type. K103N + L100I had a greater reduction in fitness and was less fit than K103N + V108I and K103N + P225H. The fitness defect of K103N + L100I relative to K103N was completely compensated for by the addition of the nucleoside resistance mutation L74V. In the presence of efavirenz, L100I was less fit than K103N, and K103N + L100I was more fit than K103N + V108I. Our studies suggest the primary driving force behind the selection of secondary efavirenz resistance mutations is the acquisition of higher levels of drug resistance, but the specific secondary mutations to emerge are those with the least cost in terms of replication efficiency. In addition, nucleoside and NNRTI resistance mutations can interact to affect HIV replication efficiency; these interactions may influence which mutations emerge during treatment failure. These studies have important implications for the design of more durable NNRTI–nucleoside combination regimens.
HIV-1; Replication fitness; K103N; L100I; V108I; P225H; Efavirenz
Murine norovirus (MNV) is a recently discovered pathogen that has become a common contaminant of specific pathogen-free mouse colonies. MNV-1 induces a robust interferon-β response and causes histopathology in some mouse strains, suggesting that it may impact other mouse models of infection. Despite many concerns about MNV-1 contamination, there is little information about its impact on immune responses to other infections. This study addresses whether MNV-1 infection has an effect on a model of murine cytomegalovirus (MCMV) infection. Exposure to MNV-1 resulted in a decreased CD8 T cell response to immunodominant MCMV epitopes in both BALB/c and C57BL/6 mice. However, MNV-1 did not impact MCMV titers in either mouse strain, nor did it stimulate reactivation of latent MCMV. These data suggest that while MNV-1 has a mild impact on the immune response to MCMV, it is not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model.
The ability of HIV to establish latent infection in CD4+ lymphocytes represents a major barrier to the eradication of HIV. It is not clear what mechanisms favor latent over productive infection, but prior studies have suggested a role for the viral transcription factor Tat or its RNA target, TAR. Using samples from five individuals who were started on ART within 6 months of infection and achieved a viral load <50 (suppressed), we isolated one- and two-exon tat RNA from HIV propagated ex vivo from baseline plasma and from co-cultures of CD4+ T cells obtained at baseline and suppressed time points. Compared to virus from the baseline plasma (mostly from productively-infected CD4+ T cells), virus from the baseline and suppressed co-cultures (mostly from latently-infected cells) had more Tat variants with impaired transactivation activity. These findings suggest that impaired activity in the Tat-TAR axis may contribute to the establishment of latent infection in CD4+ T cells.
HIV; latency; Tat; Transcription; Transactivation; TAR; CD4; Early infection
Mother-to-child transmission (MTCT) of HIV-1 provides a model for studying the role of passively acquired antibodies in preventing HIV infection. We determined the titers of neutralizing antibodies (NAbs) against six primary isolates of clades B and CRF01_AE in sera from 45 transmitting and 45 nontransmitting mothers matched for the main independent factors associated with MTCT in Thailand. A lower risk of MTCT, particularly for intrapartum transmission, was associated only with higher NAb titers against the CRF01_AE strain, MBA. The envelope glycoprotein of this strain showed an unusually long V2 domain of 63 amino acids, encoding six potential N-linked glycosylation sites. We provided experimental data indicating that the extended V2 domain contributed to the higher level of resistance to neutralization by mothers' sera in this strain. Taken together the data suggest that some primary isolates with specific properties may be useful indicators for identifying protective antibodies.
HIV; mother-to-child transmission; neutralizing antibodies