Protein 2A is not required for Theiler's virus replication.

Nonpolar mutations were introduced into all 12 regions of the genome of Theiler's murine encephalomyelitis virus. In agreement with data previously reported for other picornaviruses, mutations in regions 2B, 2C, 3A, 3B, 3C, and 3D totally abrogated viral RNA replication. Viruses with deletions in each of the capsid proteins retained RNA replication proficiency, although they were unable to propagate from cell to cell. As reported previously, mutations in the leader protein did not impair RNA replication or virus production in BHK-21 cells. Surprisingly, region 2A also appeared to be dispensable for the replication process. Indeed, up to 77 of the 133 amino acids of 2A could be deleted without significantly affecting RNA replication. 2A mutant viruses had only a slow cytopathic effect for BHK-21 cells and were totally avirulent for mice. As was the case for mutants lacking the leader protein, viruses with deletions in 2A propagated in BHK-21 cells, but their propagation was highly restricted in L929 cells.     

Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.     

Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.

We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.     

Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2'-OH methylations of the viral RNA cap (GpppA-RNA-->m(7)GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5'-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5'-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5' stem-loop of the genomic RNA.     

Impact of Potential Permissive Neuraminidase Mutations on Viral Fitness of the H275Y Oseltamivir-Resistant Influenza A(H1N1)pdm09 Virus In Vitro,in Mice and in Ferrets

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) generally compromise the fitness of influenza viruses. The only NAI-resistant virus that widely spread in the population, the A/Brisbane/59/2007 (H1N1) strain, contained permissive mutations that restored the detrimental effect caused by the H275Y change. Computational analysis predicted other permissive NA mutations for A(H1N1)pdm09 viruses. Here, we investigated the effect of T289M and N369K mutations on the viral fitness of the A(H1N1)pdm09 H275Y variant. Recombinant wild-type (WT) A(H1N1)pdm09 and the H275Y, H275Y/T289M, H275Y/N369K, and H275Y/V241I/N369K (a natural variant) NA mutants were generated by reverse genetics. Replication kinetics were performed by using ST6GalI-MDCK cells. Virulence was assessed in C57BL/6 mice, and contact transmission was evaluated in ferrets. The H275Y mutation significantly reduced viral titers during the first 12 to 36 h postinfection (p.i.) in vitro. Nevertheless, the WT and H275Y viruses induced comparable mortality rates, weight loss, and lung titers in mice. The T289M mutation eliminated the detrimental effect caused by the H275Y change in vitro while causing greater weight loss and mortality in mice, with significantly higher lung viral titers on days 3 and 6 p.i. than with the H275Y mutant. In index ferrets, the WT, H275Y, H275Y/T289M, and H275Y/V241I/N369K recombinants induced comparable fever, weight loss, and nasal wash viral titers. All tested viruses were transmitted at comparable rates in contact ferrets, with the H275Y/V241I/N369K recombinant demonstrating higher nasal wash viral titers than the H275Y mutant. Permissive mutations may enhance the fitness of A(H1N1)pdm09 H275Y viruses in vitro and in vivo. The emergence of such variants should be carefully monitored.     

PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.     

Fitness alteration of foot-and-mouth disease virus mutants: measurement of adaptability of viral quasispecies.

We document the rapid alteration of fitness of two foot-and-mouth disease virus (FMDV) mutants resistant to a neutralizing monoclonal antibody. Both mutants showed a selective disadvantage in BHK-21 cells when passaged in competition with their parental FMDV. Upon repeated replication of the mutants alone, they acquired a selective advantage over the parental FMDV and fixed additional genomic substitutions without reversion of the monoclonal antibody-resistant phenotype. Thus, variants that were previously kept at low frequency in the mutant spectrum of a viral quasispecies rapidly became the master sequence of a new genomic distribution and dominated the viral population.     

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.     

Requirement for the amino-terminal domain of sindbis virus nsP4 during virus infection

The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG((8-10))VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. Three changes in nsP1, I351 to V, I388 to V, or the previously identified change, N374 to H (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8641-8649, 2002), suppressed the minus-strand synthetic defect. A direct reversion back to G at position 8 reduced the RNA synthesis defect of the GPG((8-10))VAV virus. These results imply that nsP4's amino-terminal domain participates in distinct interactions with other nsPs in the context of differentially functioning RNA synthetic complexes, and flexibility in this domain is important for viral RNA synthesis. Additionally, the inability of the mutant viruses to efficiently inhibit host protein synthesis suggests a role for nsP4 in the regulation of host cell gene expression.     

Assessing in vivo mutation frequencies and creating a high-resolution genome-wide map of fitness costs of Hepatitis C virus

Like many viruses, Hepatitis C Virus (HCV) has a high mutation rate, which helps the virus adapt quickly, but mutations come with fitness costs. Fitness costs can be studied by different approaches, such as experimental or frequency-based approaches. The frequency-based approach is particularly useful to estimate in vivo fitness costs, but this approach works best with deep sequencing data from many hosts are. In this study, we applied the frequency-based approach to a large dataset of 195 patients and estimated the fitness costs of mutations at 7957 sites along the HCV genome. We used beta regression and random forest models to better understand how different factors influenced fitness costs. Our results revealed that costs of nonsynonymous mutations were three times higher than those of synonymous mutations, and mutations at nucleotides A or T had higher costs than those at C or G. Genome location had a modest effect, with lower costs for mutations in HVR1 and higher costs for mutations in Core and NS5B. Resistance mutations were, on average, costlier than other mutations. Our results show that in vivo fitness costs of mutations can be site and virus specific, reinforcing the utility of constructing in vivo fitness cost maps of viral genomes.     

Alteration of encephalomyocarditis virus pathogenicity due to a mutation at position 100 of VP1

Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis, myocarditis and diabetes in its natural host, the mouse. In this study, we generated four cDNA clones with a point mutation at position 100 of VP1. The amino acids isoleucine, alanine, serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis, and viable viruses were rescued. Although all mutants and wild-type viruses display different plaque morphologies, they replicate comparably in BHK-21 cells. The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice. The results showed that the isoleucine- (T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality, lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus. These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection, and its mutation alters the pathogenicity of viral infection in mice.     

Resuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virus

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37 degrees C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28 degrees C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.     

Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco Etch virus

Little is known about the fitness and virulence consequences of single-nucleotide substitutions in RNA viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. Here we used site-directed mutagenesis to create a collection of 66 clones of Tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. Competition experiments between each mutant and the ancestral nonmutated clone were performed in planta to quantitatively assess the relative fitness of each mutant genotype. Among all mutations, 40.9% were lethal, and among the viable ones, 36.4% were significantly deleterious and 22.7% neutral. Not a single case of beneficial effects was observed within the level of resolution of our measures. On average, the fitness of a genotype carrying a deleterious but viable mutation was 49% smaller than that for its unmutated progenitor. Deleterious mutational effects conformed to a beta probability distribution. The virulence of a subset of viable mutants was assessed as the reduction in the number of viable seeds produced by infected plants. Mutational effects on virulence ranged between 17% reductions and 24.4% increases. Interestingly, the only mutations showing a significant effect on virulence were hypervirulent. Competitive fitness and virulence were uncorrelated traits.     

Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C₃Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C₃Arg/85 infection. Infection of BHK-Rb cells with C₃Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C₃-Rb. The selected C₃-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9→Ala in VP3 and either Gly-110→Arg or His-108→Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C₃Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.     

The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation

Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation ‘freezes’ IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.     

Neuroblastoma Cell Membranes: Specificity for Cell Fusion Mediated by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Abstract: Temperature-sensitive mutant G3 1 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nucleiin polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.     

Mutations in the 5’ NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.     

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.